Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2018-02-23T17:32:36Z No. of bitstreams: 2 Jean_Debastiani2017.pdf: 2133266 bytes, checksum: 2c24ca5fdccdfabea585b210fcf8110f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-02-23T17:32:36Z (GMT). No. of bitstreams: 2 Jean_Debastiani2017.pdf: 2133266 bytes, checksum: 2c24ca5fdccdfabea585b210fcf8110f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-11 / Lesions in the peripheral nervous system imply active in voluntary motor function, leading to increased morbidity, incapacities in work activities and daily life, with consequent increase in costs for the public health system. Effective treatments research, studies have shown that physical and efficient exercise in functional recovery after the development of the nervous system, which is not a process of nerve regeneration. In addition, several products are being investigated with no intention to aid recovery, there are no cases of sericin silk protein, studies indicate its action in the healing process, contributing in the tissue regeneration in chronic lesions. The aim of this study was to verify the role of sericin associated with a swimming exercise protocol, with overload, without repair of the sciatic nerve of Wistar rats after nerve compression. For this purpose, 80 animals were divided into five groups of 16 animals each, being: control (CON), Injury (LES), injury-sericin (LSER), injury-swimming (LNAT) and injury-sericin-swimming (LSRNT). The animals of the LES, LSER, LNAT and LSRNT groups were submitted to compression of the sciatic nerve, however LSER and LSRNT received an application of hydrolyzed sericin protein directly on a nerve lesion at the time of surgery. The LNAT and LSRNT groups underwent swimming exercise with a load of 10% of the body weight of the animal, for cycles of five days of training and two of rest, of 1 or 3 cycles, according to the euthanasia times, and with progression of time, being in the first cycle 15 minutes of exercise, in the second 20 minutes and 25 minutes in the third cycle. During the experimental period, animals as well as nociception, by the digital "Von Frey" filament test and function analysis by means of the functional index of the ischiatic. Euthanasia was performed in two stages, the sample being euthanized on the 8th postoperative day, and the remainder on the 22nd day. Two fragments of the nerve, distal to the nerve injury, were collected and prepared for light microscopy analysis, stained with hematoxylin-eosin and pre-stained with carbon tetraoxide, being observed as morphological and morphometric characteristics of the nerve fiber. The results were analyzed statistically as their normality and one-way ANOVA was performed, with LSD post-test. Regarding the euthanized animals on the eighth day, a sericin applied directly on the sciatic nerve lesion, presented proinflammatory characteristics, and an isolated swimming showed an improvement of hypernociception in relation to the other interventions. There were no signs of acceleration of the nerve repair process on the eighth day after injury. In relation to the euthanized animals on the 22nd day, an association of sericin and swimming treatment evidenced a regulating effect of the serinin pro-inflammatory action by the resisted swimming exercise, and an isolated sericin caused exacerbation of the inflammatory process and resistance swimming exercise, It showed good evolution to the nociceptive threshold and allodynia. / Lesões no sistema nervoso periférico implicam em alterações na função motora voluntária, levando ao aumento da morbidade, incapacidades nas atividades laborais e de vida diária, com consequente aumento dos custos para o sistema de saúde público. Na busca de tratamentos efetivos, estudos têm demonstrado que o exercício físico é eficiente na recuperação funcional após lesão do nervo isquiático, sendo que a natação pode produzir a aceleração no processo de regeneração nervosa. Além disso, diversos produtos vêm sendo investigados no intuito de auxiliar a recuperação, no caso da proteína da seda sericina, estudos apontam sua ação no processo de cicatrização, contribuindo na regeneração tecidual em lesões crônicas. Observados estes aspectos, esta pesquisa teve por objetivo verificar o papel da sericina associada a um protocolo de exercício de natação, com sobrecarga, no reparo do nervo isquiático de ratos Wistar após compressão nervosa. Para tanto, foram utilizados 80 animais divididos em cinco grupos de 16 animais cada, sendo: controle (CON), lesão (LES), lesão-sericina (LSER), lesão-natação (LNAT) e lesão-sericina-natação (LSRNT). Os animais dos grupos LES, LSER, LNAT e LSRNT foram submetidos à compressão do nervo isquiático, porém somente LSER e LSRNT receberam a aplicação de proteína sericina hidrolisada diretamente sobre a lesão nervosa no momento da cirurgia. Os grupos LNAT e LSRNT realizaram exercício de natação com carga de 10% do peso corporal do animal, por ciclos de cinco dias de treino e dois de descanso, de 1 ou 3 ciclos, de acordo com os tempos de eutanásia, e com progressão de tempo, sendo no primeiro ciclo 15 minutos de exercício, no segundo 20 minutos e 25 minutos no terceiro ciclo. Durante o período experimental, os animais foram avaliados quanto à nocicepção, pelo teste de filamento tipo “Von Frey” digital e análise de função por meio do índice funcional do isquiático. A eutanásia foi realizada em duas etapas, sendo metade da amostra eutanasiada no 8º dia de pós-operatório, e o restante no 22º dia. Dois fragmentos do nervo, distais à lesão nervosa, foram coletados e preparados para analise em microscopia de luz, corado com hematoxilina-eosina e pré-corado com tetróxido de ósmio respectivamente, sendo observadas as características morfológicas e morfometricas da fibra nervosa. Os resultados obtidos foram analisados estatisticamente quanto sua normalidade e realizado ANOVA unidirecional, com pós-teste de LSD. Em relação aos animais eutanasiados no oitavo dia, a sericina aplicada diretamente sobre a lesão nervosa isquiática, apresentou características pró-inflamatórias, já a natação isolada mostrou melhora da hipernocicepção em relação às demais intervenções. Não houve sinais de aceleração do processo de reparo nervoso no oitavo dia após a lesão. Já em relação aos animais eutanasiados no 22º dia, a associação do tratamento de sericina e natação evidenciou um efeito regulador da ação pró-inflamatória da sericina pelo exercício resistido de natação, já a sericina isolada provocou exacerbação do processo inflamatório e o exercício de natação resistido, mostrou boa evolução quanto ao limiar nociceptivo e alodinia.

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2018-02-23T19:34:28Z No. of bitstreams: 2 André_Santana2017.pdf: 2268024 bytes, checksum: d4eee1d456e910253f049c57a48abf85 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-02-23T19:34:28Z (GMT). No. of bitstreams: 2 André_Santana2017.pdf: 2268024 bytes, checksum: d4eee1d456e910253f049c57a48abf85 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-04 / Peripheral nerve damage causes a number of morphological changes, resulting in functional, nerve and muscle complications. There are several therapeutic measures applied in rehabilitation, such as physical exercise in the aquatic environment, which has been extensively studied in terms of functional improvement, although its regenerative potential needs more evidence. Also, it is important to search for substances with therapeutic potential and that can be used in association with physical exercise, in order to intensify recovery. The protein biopolymer, sericin, obtained from the cocoon of the silkworm (Bombyx mori), presents a series of important regenerative pharmacological effects, with cicatrizant action in the treatment of burns, improvement in the aerobic performance and the oxidation of fat at rest. In this sense, the objective of this study was to analyze the effect of sericin associated or not to physical swimming exercise on the muscular recovery of Wistar rats, submitted to sciatic nerve injury. The experiment was carried out in a sample composed of 40 animals, 10 ± 2 weeks old, randomly divided into five groups: Ct: control; Ls: injury; Being: injury + sericin; Nat: injury + swimming; Ser + Nat: injury + sericina + physical exercise. The animals were anesthetized and submitted to compression injury of the right sciatic nerve. Immediately after the nerve compression, a dose of 100 μL of hydrolyzed sericin was applied to the injured nerve in Ser and Ser + Nat animals. On the other hand, the animals of the Nat and Ser + Nat groups, 72 hours after the injury, were treated with resisted physical exercise of swimming, with overload of 10% of body weight, during three weeks, five days a week. The animals performed fifteen minutes of swimming in the first week, 20 minutes in the second and 25 minutes in the third week. During the treatment, the grip strength of the right pelvic limb of all animals was evaluated. At the end of the experimental period, the animals were anesthetized for dissection and collection of the plantar muscle, and the proximal part was processed and analyzed histomorphologically, and the distal for histoenzymological analysis. Regarding the functional data of muscular strength of grip, morphology and morphometry of the 8 neuromuscular junctions, no significant influence on the neuromuscular regeneration process was observed. The same occurred with the musculoskeletal properties, which did not suffer significant changes in the association of sericin and swimming. Although physical swimming exercise alone was efficient in maintaining the intramuscular conjunctiva, the association with sericin was not able to alter the plantar muscle phenotype, although experimental axonotmosis did so. / Lesões nervosas periféricas causam uma série de alterações morfológicas, resultando em complicações funcionais, nervosas e musculares. Várias são as medidas terapêuticas aplicadas na reabilitação, como, por exemplo, o exercício físico em meio aquático, que vem sendo amplamente estudado no que diz respoieto à melhora funcional, embora seu potencial regenerativo necessite de maiores comprovações. Ainda, é importante a busca de substâncias com potencial terapêutico e que possam ser utilizadas em associação ao exercício físico, de forma a intensificar a recuperação. O biopolímero protéico sericina, obtido do casulo do bicho-da-seda (Bombyx mori), apresenta uma série de efeitos farmacológicos regenerativos importantes, com ação cicatrizante no tratamento de queimaduras, melhora no desempenho aeróbico e na oxidação de gordura em repouso. Nesse sentido, o objetivo deste estudo foi analisar o efeito da sericina associada ou não ao exercício físico de natação sobre a recuperação muscular de ratos Wistar, submetidos à lesão nervosa isquiática. O experimento foi desenvolvido numa amostra composta por 40 animais, com 10±2 semanas de idade, separados aleatoriamente em cinco grupos: Ct: controle; Ls: lesão; Ser: lesão + sericina; Nat: lesão + natação; Ser+Nat: lesão + sericina + exercício físico. Os animais foram anestesiados e submetidos à lesão por compressão do nervo isquiático direito. Imediatamente após a compressão nervosa, nos animais dos grupos Ser e Ser+Nat, foram aplicadas uma dose de 100 μL de sericina hidrolisada sobre o nervo lesionado. Já os animais dos grupos Nat e Ser+Nat, 72 horas após a lesão, foram tratados com exercício físico resistido de natação, com sobrecarga de 10% da massa corporal, durante três semanas, cinco dias por semana. Os animais realizaram quinze minutos de natação na primeira semana, 20 minutos na segunda e 25 minutos na terceira semana. No decorrer do tratamento, avaliou-se a força de preensão do membro pélvico direito de todos os animais. Ao término do período experimental, os animais foram anestesiados para dissecação e coleta do músculo plantar, sendo que a parte proximal foi processada e analisada histomorfologicamente, e a distal para análise histoenzimológica. Com relação aos dados funcionais de força muscular de preensão, morfologia e morfometria das junções neuromusculares, não foi observado influência significativa sobre o processo de regeneração neuromuscular. O mesmo ocorreu com as propriedades músculo esqueléticas, que não sofreram alterações significativas na associação da sericina e da natação. Embora o exercício físico de natação sozinho tenha sido eficiente na manutenção do conjuntivo intramuscular, a associação com sericina não foi capaz de alterar o fenótipo do músculo plantar, embora a axonotmese experimental o tenha feito.

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2018-03-06T17:41:31Z No. of bitstreams: 2 Andre_Santana2017.pdf: 2268027 bytes, checksum: 9d06e9ab0f5b41d9bdfbfbafcbba9200 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-03-06T17:41:31Z (GMT). No. of bitstreams: 2 Andre_Santana2017.pdf: 2268027 bytes, checksum: 9d06e9ab0f5b41d9bdfbfbafcbba9200 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-04 / Peripheral nerve damage causes a number of morphological changes, resulting in functional, nerve and muscle complications. There are several therapeutic measures applied in rehabilitation, such as physical exercise in the aquatic environment, which has been extensively studied in terms of functional improvement, although its regenerative potential needs more evidence. Also, it is important to search for substances with therapeutic potential and that can be used in association with physical exercise, in order to intensify recovery. The protein biopolymer, sericin, obtained from the cocoon of the silkworm (Bombyx mori), presents a series of important regenerative pharmacological effects, with cicatrizant action in the treatment of burns, improvement in the aerobic performance and the oxidation of fat at rest. In this sense, the objective of this study was to analyze the effect of sericin associated or not to physical swimming exercise on the muscular recovery of Wistar rats, submitted to sciatic nerve injury. The experiment was carried out in a sample composed of 40 animals, 10 ± 2 weeks old, randomly divided into five groups: Ct: control; Ls: injury; Being: injury + sericin; Nat: injury + swimming; Ser + Nat: injury + sericina + physical exercise. The animals were anesthetized and submitted to compression injury of the right sciatic nerve. Immediately after the nerve compression, a dose of 100 μL of hydrolyzed sericin was applied to the injured nerve in Ser and Ser + Nat animals. On the other hand, the animals of the Nat and Ser + Nat groups, 72 hours after the injury, were treated with resisted physical exercise of swimming, with overload of 10% of body weight, during three weeks, five days a week. The animals performed fifteen minutes of swimming in the first week, 20 minutes in the second and 25 minutes in the third week. During the treatment, the grip strength of the right pelvic limb of all animals was evaluated. At the end of the experimental period, the animals were anesthetized for dissection and collection of the plantar muscle, and the proximal part was processed and analyzed histomorphologically, and the distal for histoenzymological analysis. Regarding the functional data of muscular strength of grip, morphology and morphometry of the 8 neuromuscular junctions, no significant influence on the neuromuscular regeneration process was observed. The same occurred with the musculoskeletal properties, which did not suffer significant changes in the association of sericin and swimming. Although physical swimming exercise alone was efficient in maintaining the intramuscular conjunctiva, the association with sericin was not able to alter the plantar muscle phenotype, although experimental axonotmosis did so / Lesões nervosas periféricas causam uma série de alterações morfológicas, resultando em complicações funcionais, nervosas e musculares. Várias são as medidas terapêuticas aplicadas na reabilitação, como, por exemplo, o exercício físico em meio aquático, que vem sendo amplamente estudado no que diz respoieto à melhora funcional, embora seu potencial regenerativo necessite de maiores comprovações. Ainda, é importante a busca de substâncias com potencial terapêutico e que possam ser utilizadas em associação ao exercício físico, de forma a intensificar a recuperação. O biopolímero protéico sericina, obtido do casulo do bicho-da-seda (Bombyx mori), apresenta uma série de efeitos farmacológicos regenerativos importantes, com ação cicatrizante no tratamento de queimaduras, melhora no desempenho aeróbico e na oxidação de gordura em repouso. Nesse sentido, o objetivo deste estudo foi analisar o efeito da sericina associada ou não ao exercício físico de natação sobre a recuperação muscular de ratos Wistar, submetidos à lesão nervosa isquiática. O experimento foi desenvolvido numa amostra composta por 40 animais, com 10±2 semanas de idade, separados aleatoriamente em cinco grupos: Ct: controle; Ls: lesão; Ser: lesão + sericina; Nat: lesão + natação; Ser+Nat: lesão + sericina + exercício físico. Os animais foram anestesiados e submetidos à lesão por compressão do nervo isquiático direito. Imediatamente após a compressão nervosa, nos animais dos grupos Ser e Ser+Nat, foram aplicadas uma dose de 100 μL de sericina hidrolisada sobre o nervo lesionado. Já os animais dos grupos Nat e Ser+Nat, 72 horas após a lesão, foram tratados com exercício físico resistido de natação, com sobrecarga de 10% da massa corporal, durante três semanas, cinco dias por semana. Os animais realizaram quinze minutos de natação na primeira semana, 20 minutos na segunda e 25 minutos na terceira semana. No decorrer do tratamento, avaliou-se a força de preensão do membro pélvico direito de todos os animais. Ao término do período experimental, os animais foram anestesiados para dissecação e coleta do músculo plantar, sendo que a parte proximal foi processada e analisada histomorfologicamente, e a distal para análise histoenzimológica. Com relação aos dados funcionais de força muscular de preensão, morfologia e morfometria das junções neuromusculares, não foi observado influência significativa sobre o processo de regeneração neuromuscular. O mesmo ocorreu com as propriedades músculo esqueléticas, que não sofreram alterações significativas na associação da sericina e da natação. Embora o exercício físico de natação sozinho tenha sido eficiente na manutenção do conjuntivo intramuscular, a associação com sericina não foi capaz de alterar o fenótipo do músculo plantar, embora a axonotmese experimental o tenha feito.

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