No description available.

Skin is an organ with a complex structure which plays a crucial role in thebody’s defence against external threats and in maintaining major homeostatic functions. The need for in vitro models that mimic the in vivo milieu is therefore high and relevant with various applications including, among others, penetration, absorption, and toxicity studies. In this context, the choice of the biomaterial that will provide a 3D scaffold to the cultured cells is defining the model’s success. The FN-4RepCT silk is here suggested as a potent biomaterial for skin tissue engineering applications. This recombinantly produced spider silk protein (FN-4RepCT), which can self-assemble into fibrils, creates a robust and elastic matrice with high bioactivity, due to its functionalization with the fibronectin derived RGD-containing peptide. Hence it overcomes the drawbacks of other available biomaterials either synthetic or based on animal derived proteins. Additionally, the FN-4RepCT silk protein can be cast in various 3D formats, two of which are utilized within this project. We herein present a bilayered skin tissue equivalent supported by the FN-4RepCT silk. This is constructed by the combination of a foam format, integrated with dermal fibroblasts and endothelial cells, and a membrane format supporting epidermal keratinocytes. As a result, a vascularized dermal layer that contains ECM components (Collagen I, Collagen III, and Elastin) is constructed and attached to an epidermal layer of differentiated keratinocytes.The protocol presented in this project offers a successful method of evenly integrating cells in the FN-4RepCT silk scaffold, while preserving their ability to resume some of their major in vivo functions like proliferation, ECM secretion, construction of vascular networks, and differentiation. The obtained results were evaluated with immunofluorescence stainings of various markers of interest and further analysed, when necessary, with image processing tools. The results that ensued from the herein presented protocol strongly suggest that the FN-4RepCT silk is a promising biomaterial for skin tissue engineering applications.

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Development of methods for studying drug delivery systems is of great significance for the improvement of topical formulations. Active compounds for topical drug delivery are often formulated into gels and creams, that can be applied onto skin surfaces. It is important to know the extent of the permeability of the active compounds, in order to determine the medical effect. This study examines the possibilities of using an animal-free skin equivalent for penetration and permeation experiments, i.e. a silk scaffold integrated with viable human dermaland epidermal cells. Mammalian cell culturing together with silkconstruct formation, constituted the upstream bioprocess and acquisition of the skin equivalents. Permeability of fluorescently labelled Bovine Serum Albumin and Sodium Fluorescein salt was assessed, using a Franz- cell setup incorporated with the skin equivalents. Furthermore, fluorescence analysis and SDS-PAGE was performed on the collected samples, along with cryosectioning and image analysis of the skin equivalents. The results indicate variations in tissue integrity, leading to both high and low permeability. Fluorescence intensity can be correlated with the amount of sample liquid passing through. The model is still under development, hence more research is needed to draw a conclusion regarding the cellular composition of the skin equivalents, and how it influences permeability. / NextBioForm

Bröstcancer är den vanligaste formen av cancer som drabbar kvinnor. Det är en heterogen och komplex sjukdom som består av flera undergrupper, var och en med distinkt morfologi och kliniska implikationer [1]. För att modellera och studera cellbiologi, vävnadsmorfologi, molekylära mekanismer och läkemedels effekter används cellkulturer [2]. Idag är tvådimensionella (2D) modeller fortfarande den mest använda metoden för att odla celler in vitro [3]. En nackdel med 2D-modeller är att mikromiljön i dessa modeller inte imiterar in vivo strukturen av tumörer och vävnader, då de saknar tre dimensionella (3D) cell-cell och cellextracellulär matrix (ECM) interaktioner [2]. På grund av nackdelarna med 2D-modeller, har 3D-modeller blivit mer intressanta som alternativ för att lösa behovet av en pålitlig preklinisk modell för läkemedelstestning och för studier av cancerbiologi. För att utveckla ett redskap som är relevant för cancerforskning etablerar professor My Hedhammars laboratorium en 3D-modell av bröstcancer. I en sådan ny modell används Biosilk som byggnadsställning för att odla odödliga cellinjer som är representativa för de tre huvudklasserna av bröstcancer (i.e. MCF-7 (luminal-lik), SKBR-3 (HER2-överuttryckt) och MDAMB- 231 (trippel-negativ)). Eftersom transkriptions signaturer kan användas för att klassificera och studera bröstcancer är det viktigt att undersöka om och hur tillväxt i 3D-Biosilk kan påverka genuttrycksprofiler. Hypotesen som testades i denna studie var om cellkulturer i 3DBiosilk kan ha signifikanta skillnader i uttryck av biomarkörer, relevanta för bröstcancerbiologi, vid jämförelse av samma cellinje kultiverad i 2D. För att testa detta utvärderades kvalitén och reproducerbarheten av 3D-Biosilk konstruktionen med hjälp av olika kvalitetstester. Strukturen granskades med brightfield mikroskopi, arean av konstruktionen mättes med ImageJ, infärgning med phalloidin bekräftade cellnärvaro och cellvidhäftning till modellen. Alamar blue utfördes för att bedöma den cellulära metaboliska aktiviteten i modellen. Förändringarna av målgenernas genuttryck undersöktes med kvantitativ omvänd transkription PCR (RT-qPCR) och detta påvisade en statistiskt signifikant skillnad i genuttrycket beroende på om cellerna odlats i 2D- eller 3D-Biosilk modeller. I cellinje MDA-MB-231 hittades tre gener, i cellinje SKBR-3 hittades två gener och i cellinje MCF-7 hittades fyra gener. Genuttrycket för en av dessa gener i cellinje MCF-7, som var kultiverad i 3D-Biosilk, var nedreglerad (i.e. ZO-1). Detta kunde valideras på proteinnivå med immunofluorescens. Sammanfattningsvis, celler odlade i 3D-Biosilk visar på en mer aggressiv fenotyp. / Breast cancer is the most common cancer among women. It is a heterogenous and complex disease composed of several subtypes, each with distinct morphological and clinical implications [1]. To model and study cell biology, tissue morphology, molecular mechanisms and drug actions, cell cultures are canonically used [2]. Today two-dimensional (2D) models are still widely the preferred method for culturing cells in vitro [3]. A drawback with 2D models is that the microenvironment in these models does not mimic the in vivo structure of tumors and tissues, lacking three-dimensional (3D) cell-cell and cell-extracellular matrix (ECM) interactions [2]. Due to the disadvantages of 2D models, 3D cultures have become an increasingly interesting alternative to solve the need for a reliable preclinical model for drug testing and the study of cancer biology. To develop a relevant tool for cancer research, the laboratory of professor My Hedhammar is currently establishing a 3D model of breast cancer. In such novel model, Biosilk is used as scaffold to grow immortalized cell lines representative of the three major classes of breast cancer (i.e. MCF-7 (luminal-like), SKBR-3 (HER2-overexpression) and MDA-MB-231 (triplenegative)). Since transcriptional signatures can be used to classify and study breast cancers, it is important to investigate if and how growth in 3D-Biosilk can impact gene expression profiles. The hypothesis tested in this study was that cells cultured in 3D-Biosilk have differences in expression of biomarkers relevant to breast cancer biology, when compared to the same cell lines cultured in 2D. To examine this, 3D-Biosilk models were created and evaluated to ensure their quality and reproducibility, for instance, the scaffold structure was monitored by brightfield microscopy, the construct’s area was measured with ImageJ, staining with phalloidin confirmed the presence of cells as well as their attachment to the construct, and Alamar blue was used to assess the cellular metabolic activity. Differences in gene expression of target genes were investigated using reverse transcription quantitative PCR (RTqPCR), which revealed statistically significant changes depending on whether the cells were cultivated in 2D or a 3D-Biosilk model. For cell line MDA-MB-231 three genes were found, for SKBR-3 two genes were found and for MCF-7 four genes were found. The expression of one gene which was found downregulated in MCF-7 cultured in 3D-Biosilk (i.e. ZO-1) was validated at protein level by immunofluorescence. In conclusion, cultivating cells in 3D-Biosilk indicates a more aggressive phenotype.

No description available.

Spider silk has outstanding mechanical properties, displaying high tensile strength and extensibility. The unique combination of strength and great extensibility make it one of the toughest materials in the world. Of the seven different spider silks, dragline silk, the lifeline silk of the spider, represents one of the most renowned fiber types that has extraordinary properties. As a result, many labs across the globe are racing to manufacture synthetic dragline silk fibers. With the production of synthetic dragline silk fibers, there are unlimited commercial applications. In this study, we developed several codon-optimized MaSp1 minifibroin constructs for recombinant protein expression in bacteria. These recombinant MaSp1 minifibroin constructs were engineered to contain the N-terminal domain (NTD), different copies of internal block repeats (ranging from 2 to 64 copies of 35 amino acid blocks), and the C-terminal domain (CTD). The NTD and CTDs were derived from the natural cDNA sequences of black widow spiders, while the internal block repeats were generated from synthetic DNA fragments that were codon-optimized for expression in Escherichia coli . Different numbers of internal block repeats were created using a specialized seamless cloning strategy. By applying this seamless cloning strategy, we successfully multimerized MaSp1 block repeats that approach the natural fibroin size. Moreover, through the construction of a customized NTD-CTD spidroin construct, multimerized block repeats from any fibroin can be rapidly inserted to facilitate minifibroin protein expression in bacteria. Overall, this strategy as well as the created vectors, should help advance the silk community in the production of synthetic silk fibers that have properties that more closely resemble natural fibers.

No description available.

Spider silk is one of the most fascinating materials found in nature. Besides its properties like biodegradability, low immunoreactivity, and biocompatibility, especially the mechanical properties outperforming today’s artificial high-tech materials like Kevlar® are of great interest in biomedicine or material science. Spider silk comprises highly repetitive amino acid sequence motives, whose structure is accepted to be responsible for the extraordinary properties of spider silk. Typically, hydrophilic sequence motives alternate with hydrophobic ones making spider silk proteins resemble block copolymers. Additionally, the simple amino acid sequence and the possibility to form fibrillar structures are common characteristics of spider silk proteins as well as intrinsically disordered proteins (IDP) or protein regions (IDR). Both are suspected of being involved in the development of certain neurodegenerative diseases like Alzheimer´s disease. These aspects open promising possibilities of the use of spider silk proteins in nanotechnology, but also as model systems for the fibrillization processes of IDPs and IDRs, which are still unresolved today. Currently, most of the research and application is focused on 1-dimensional spider silk protein fibrils and fibers or 0-dimensional spider silk particles. However, 2-dimensional spider silk protein films or porous 3-dimensional objects are highly relevant platforms with the potential for cell-supporting scaffolds, biodegradable electrolyte materials in transistors, or e.g., planar drug-eluting implant coatings. Generally, the effects of sequence-based and external influences on the self-assembly and folding of spider silk proteins have not yet been fully elucidated in all of these various dimensional spider silk materials, even concerning IDP and IDR models. Thus, basic research regarding assembly and folding processes is still needed, especially in films. Particularly, 2-dimensional films allow a broad spectrum of (surface) analytical techniques, from whose outcome general structure-property relations of spider silk materials across all material dimensions can be obtained. In this work, engineered spider silk proteins, which are based on the consensus sequence motives in the spider silk fibroin (spidroin) 3 and 4 of the European garden spider Araneus diadematus (eADF4(Cx), eADF3(AQ)x, eADF3(QAQ)x) as well as blends of two short peptides with the respective aa sequence of the hydrophobic (pep-c) and hydrophilic (pep-a) part of eADF4(Cx) proteins were used. Spider silk-related proteins and peptides were dissolved in 1,1,1,3,3,3-hexafluoroisopropanol or formic acid, processed as thin films, and post-treated with methanol vapor to induce β-sheet formation. Dichroic FTIR-spectroscopy was used, a powerful tool for studying protein secondary structure formation and orientation. Proteins reveal characteristic amide bands, which are highly sensitive to the conformation of the protein backbone. In the course of this work, a set of components for the line shape analysis (LSA) of the Amide I band was developed. Therby, each component was assigned to a typical secondary structure allowing a quantitative determination of the respective portions and their structural orientation. Quantitative secondary structure portions and their orientation could be determined on this basis. Furthermore, a comprehensive study of folding and self-assembly-influencing parameters like hydrophobic and hydrophilic sequences, molecular weight, the repeating sequence motive order, the film thickness, surface topography, and the surface chemistry in engineered spider silk protein and spider silk protein-based films was carried out. In general, methanol vapor post-treatment induced the formation of β-sheet structures in all films, causing phase separation and the formation of spherical and filamentous structures. The phase separation upon post-treatment was influenced by the covalent connectivity between hydrophobic and hydrophilic sequence parts as well as the repeating sequence motives. In thin films, the increased flexibility of shorter peptides enabled the formation of multipack filaments instead of spherical structures, which were formed by higher molecular weight proteins with several inter-connected repeating sequence motives. Stamping wrinkled structures using poly(dimethylsiloxane) substrates was possible. Filamentous structures were successfully assigned to β-sheet rich structures using infrared nanospectroscopy for the first time. Further, enhanced surface hydrophobicity led to the clustering of β-sheet filaments. The β-sheet content could be controlled by the amount of hydrophobic sequences in thin films. With a higher amount of hydrophobic sequences in the proteins or blends, the β-sheet content increased until a maximum β-sheet content of around 60% was reached. Additionally, β-sheet formation could be suppressed by increasing substrate hydrophobicity or by decreasing the number of repeating sequence motives by going from protein-like folding to peptide-like self-assembly. The backfolding of proteins with covalently linked repeating sequence motives further promoted the formation of more antiparallel β-sheets. Antiparallel β-sheet formation was also favored when the portion of the hydrophilic, amorphous phase was increased. Micrometer thick films did not reveal any preferred alignment of β-sheets, while a general out-of-plane orientation of β-sheets could be obtained in all thin protein, peptide, and blend films. Z-axial orientation in films was increased by using short pep-c and pep-a peptides, higher molecular weight proteins or the deposition of monolayered films instead of thin multilayered films. Also, increased hydrophilicity of the substrate promoted the alignment of β-sheets perpendicular to the substrate surface. The folding kinetics and final domain size were found to be directly correlated. The amount of hydrophobic phase, backfolding, and increased flexibility due to low chain lengths increased the folding kinetics and led to smaller domain sizes. Thus, competing effects of backfolding and flexibility of the protein/peptide backbone could be rationalized. The film integrity and water contact angle were directly related to the β-sheet content and the molecular weight. Beyond the classical protein conformation and orientation analysis, the possibilities and limits of orientation analysis using dichroic attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were elaborated on the seemingly ideal oriented polymer model system of end-grafted poly(N,N-dimethylaminoethylmethacrylate) chains. Such a system featured a polymer brush regime in the swollen state with z-axial orientation expected similarly high as thin spider silk films after ptm. Moreover, dichroic ATR-FTIR spectroscopy is a promising analytical method for closing gaps in the defined assignment of brush regimes. In summary, general models of the structure formation and self-assembly of spider silk protein in films depending on the parameters mentioned above could be developed and set in relation to IDP/IDR self-assembly by using dichroic FTIR spectroscopy as the basic analysis method. The herein postulated models on the molecular level contribute to the understanding and development of future industrial applications of spider silk protein-based materials and the clarification of unresolved questions regarding IDP and IDR systems.:Abstract V Kurzfassung IX List of Publications XIII Publications in Trade Journals XIII Presentations and Posters XIII Contribution to Joint Publications XV List of Abbrevations XVII List of Symbols XIX List of Figures XXV List of Tables XXXIII 1 Introduction and Motivation 1 2 Theory 5 2.1 Proteins and Peptides 5 2.1.1 General Definition of Proteins and Peptides 5 2.1.2 Structure of Globular Proteins 7 2.1.3 Protein Folding 10 2.1.4 Intrinsically Disordered Proteins and Protein Regions 11 2.2 Block Copolymers 14 2.3 Spiders and Spider Silks 17 2.3.1 Classification of Spiders 17 2.3.2 The Natural Spider Silk Spinning Process 18 2.3.3 Structure of Spider Silk and Spider Silk Proteins 19 2.3.4 Structure-Property Relationships of Spider Silk 21 2.4 Infrared Spectroscopy 23 2.4.1 Basic Principles of Infrared Spectroscopy 23 2.4.2 Basic Equipment and IR-Technologies 27 2.4.3 Orientation Analysis using Dichroic FTIR Spectroscopy 32 2.4.4 Infrared Spectroscopy of Proteins and Peptides 38 2.4.5 Quantitative Analysis of TRANS- and ATR-FTIR Protein Spectra 43 2.5 Electronic Circular Dichroism 46 2.5.1 Basics Principles of Circular Dichroism 46 2.5.2 Circular Dichroism of Proteins and Polypeptides 48 2.5.3 Spectra Analysis 50 2.6 Atomic Force Microscopy 51 2.6.1 Setup of Atomic Force Microscopes 51 2.6.2 Basic Principles of Atomic Force Microscopy 52 2.6.3 AFM Operation Modes 55 3 Experimental Section 57 3.1 Materials 57 3.1.1 Chemicals 57 3.1.2 Substrates 57 3.1.3 Film Preparation 58 3.2 Analytical Methods 60 3.2.1 Dichroic FTIR Spectroscopy 60 3.2.2 Atomic Force Microscopy 64 3.2.3 Electronic Circular Dichroism 64 3.2.4 Spectroscopic Ellipsometry 64 3.2.5 Infrared Nanospectroscopy 65 3.2.6 Grazing Incident Small Angle X-Ray Scattering 66 4 Results 67 4.1 Self-Assembly of eADF4(C16) Films 67 4.1.1 Motivation 67 4.1.2 Dichroic FTIR Spectroscopy Characterization of ß-sheet Orientation in Spider Silk Films on Silicon Substrates 68 4.2 Influence of the Hydrophilic and Hydrophobic Blocks on Peptide Self-Assembly 90 4.2.1 Motivation 90 4.2.2 β-Sheet Structure Formation within Binary Blends of Two Spider Silk Related Peptides 90 4.2.3 Influence of the Hydrophilic and Hydrophobic Blocks on the Inner Morphology in Spider Silk Protein Based Blend Films 122 4.3 Influence of the Sequence Motive Repeating Number on Spider Silk Protein Folding 123 4.3.1 Motivation 123 4.3.2 Influence of Sequence Motive Repeating Number on Protein Folding in Spider Silk Protein Films 124 4.4 Influence of the Module Order on Spider Silk Protein Self-Assembly 152 4.4.1 Motivation 152 4.4.2 Secondary Structure upon Post-treatment 153 4.4.3 β-Sheet Orientation after Post-treatment 157 4.4.4 Morphology and Surface Properties 158 4.4.5 Conclusion 160 4.5 Surface Induced Changes of Spider Silk Protein Self-Assembly 161 4.5.1 Motivation 161 4.5.2 Variation of the Substrate Surface Chemistry and Topography 161 4.5.3 Influence of the Surface Topography on Protein Self-Assembly 162 4.5.4 Influence of the Surface Chemistry on Protein Self-Assembly 164 4.5.5 Conclusion 169 4.6 Chances and Limits of Dichroic ATR-FTIR Spectroscopy 170 4.6.1 Motivation 170 4.6.2 Novel Insights into Swelling and Orientation of End-Grafted PDMAEMA Chains by In-Situ ATR-FTIR Complementing In-Situ Ellipsometry 171 5 Conclusion and Outlook 197 6 References 203 7 Appendix 219 8 Danksagung 227 9 Eidesstattliche Versicherung 229

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