Keratins are important structural components of hair and skin. There has been extensive study of keratins from the health and medical perspectives, although little work has been done to date to investigate their basic physicochemical properties in the form of biomaterials. The work presented in this thesis aimed to study surface and interfacial adsorption and solution aggregation of water soluble keratin polypeptides (made available by previous work within the research group). A range of physical techniques were employed including spectroscopic ellipsometry (SE), neutron reflection (NR), dual polarisation interferometry (DPI), quartz crystal microbalance with dissipation (QCM-D), dynamic light scattering (DLS) and small-angle neutron scattering (SANS).A major technical advantage of the neutron techniques is the use of hydrogen/deuterium substitution to enhance structural resolution. This approach was explored to study the interaction of keratins with both conventional surfactants and novel biosurfactants. The work presented comprises four results chapters. The first examines and compares four widely used interfacial techniques, SE, DPI, QCM-D and NR, by studying the adsorption of C12E6 at the silicon oxide/water interface. Whilst the data exhibits a large degree of consistency in the interfacially adsorbed amount, each technique helped reveal unique structural information with a high degree of complementarity. The second results chapter reports on findings regarding the properties of keratin polypeptides in surface adsorption and solution aggregation. It was found that the keratins adsorbed strongly on the surface of water, and formed rugby-shaped nanoaggregates in solution, the size and shape of which responded to salt concentration. The third results chapter reports on the interfacial behaviour of keratin/surfactants complexes in bulk solution, with cationic DTAB and anionic SDS as model conventional surfactants. It was found that both the electrostatic and hydrophobic forces contributed strongly to the surface adsorption processes. The final results chapter reports on interactions of a coated keratin film with novel biosurfactants including rhamnolipids (R1 and R2 with 1 and 2 sugar head(s), respectively) and Mel-C. The keratin films formed were found to be exceptionally stable and reproducible below pH 8, and these films could be widely used as model keratin substrates for screening their binding with surfactants and bioactive molecules. Both rhamnolipids and Mel-C exhibited strong adsorption onto the keratin substrate and interestingly, whilst R1 exhibited a completely reversible adsorption, R2 showed only a partially reversible adsorption. Mel-C showed some degree of irreversible adsorption similar to R2 and exhibited the strongest adsorption at around pH 4-5. These results show mild interactions with the keratin substrate, but indicate that the extent of adsorption and desorption could be manipulated by surfactant structure or solution conditions. The findings presented in this thesis are fundamental in aiding the development of the use of keratin polypeptides as biomaterials, in applications such as personal care. The work is also highly relevant to the understanding of the interactions between surfactants and keratin molecules at interfaces and in solution.

The goal of this thesis is to introduce and explain technology of hydrolysis and subsequent processing of poultry feathers and make calculation of line for processing poultry feathers. This thesis also includes technological scheme of processing line, design and final evaluation of chosen processing technology.

Les mutations MKS1 sont impliquées dans un groupe de ciliopathies récessives létales, telles que le syndrome de Meckel-Gruber (MKS) et le syndrome de Joubert (JBT), caractérisées par une dysplasie rénale kystique, des anomalies du système nerveux central (encéphalocèle occipitale), une polydactylie, une dysgénésie biliaire et une fibrose hépatique. MKS1 a été localisée dans la zone de transition du cil dans de nombreux types cellulaires où elle joue un rôle essentiel pour la structure et la fonction des cils, en particulier la régulation de plusieurs voies de signalisation telles que Wnt et Shh.Dans le présent travail, nous avons identifié la fonction pré-ciliaire de MKS1 dans des cellules épithéliales. Nous avons montré que la localisation subcellulaire de MKS1 varie au cours de la maturation de l’épithelium, passant du cytosol où MKS1 co-localise avec le réseau de kératine, aux jonctions cellulaires, où elle co-colocalise avec les caténines. De plus, la translocation de MKS1 des jonctions au cytosol s'est avérée être mécano-sensible, suggérant que MKS1 participe à l'homéostasie épithéliale en stabilisant les jonctions cellulaires, via la transduction des signaux mécaniques liés à la compaction de l’épithelium.L’analyse fonctionnelle a démontré que le « knockdown » de MKS1 désorganise le réseau de kératine, et déstabilise les jonctions adhérentes des cellules épithéliales en culture, avec une diminution de la β-caténine jonctionnelle et une libération de l’E-cadhérine, l’α-caténine et la vinculine dans le cytosol. De plus, la déplétion de MKS1 entraîne une diminution notable du réseau apical d’actine, ainsi que la désorganisation de la structure épithéliale et une transition partielle vers un état mésenchymateux. Ces résultats illustrent une fonction indépendante du cil de MKS1 dans l’homéostasie épithéliale, et apporte de nouvelles hypothèses quant à son rôle et celui des filaments intermédiaires dans les processus d’organogenèse des épitheliums, en particulier la tubulogenèse, qui repose à la fois sur l’équilibre de la transition épithelium/mesenchyme et la mécanotransduction des sollicitations mécaniques durant l’embryogenèseDans le but de caractériser les partenaires de MKS1, des expériences de Co-IP et d’analyses protéomiques ont permis d’identifier l’epiplakine comme un partenaire possible de MKS1. L'Epiplakine est un cytolinker capable de lier la kératine à la membrane et à l'actine ; l’interaction de MKS1 avec l’epiplakine pourrait ainsi rendre compte de la stabilisation à la fois du réseau de kératine et des jonctions cellulaires. Des analyses complémentaires de protéomique et des études fonctionnelles devront compléter ces résultats préliminaires.Finalement, ces travaux ont également permis de révéler le rôle de MKS1 dans la stabilisation des jonctions communicantes ; la déplétion de MKS1 conduisant à une diminution de la CX43 jonctionnelle et à une altération de la fonction de communication intercellulaire dans les cellules épithéliales en culture. Ces travaux, qui constituent la première mention d’une altération possible des jonctions communicantes dans ce type de maladies, devront être approfondis pour caractériser leur impact dans les processus de tubulogenèse.En conclusion, ce travail qui a permis de révéler un rôle pré-ciliaire de MKS1 dans l'homéostasie épithéliale, apporte de nouvelles hypothèses pour l’étiologie de ces maladies, jusqu’alors considérées comme essentiellement consécutives à des défauts de transduction de la signalisation ciliaire. Il propose également de nouveaux mécanismes pour rendre compte des anomalies du développement hépatique, telles que la dysgénésie des voies biliaires, et plus largement des processus de tubulogenèse impliqués dans le développement de nombreux organes. / MKS1 mutations are involved in a group of lethal recessive ciliopathies, such as Meckel-Gruber syndrome (MKS) and Joubert's syndrome (JBT), characterized by cystic renal dysplasia, central nervous system abnormalities (occipital encephalocele) , polydactyly, biliary dysgenesis and hepatic fibrosis. MKS1 has been located in the transition zone of the cilia in many cell types where it plays an essential role in the cilia structure and function, in particular in the regulation of signaling pathways such as Wnt and Shh.In the present work, we have identified the preciliary function of MKS1 in epithelial cells. We have shown that the subcellular localization of MKS1 varies during the maturation of the epithelium, from the cytosol where MKS1 co-localizes with the keratin network, to the cell junctions, where it co-localizes with the catenins. In addition, the MKS1 translocation to cytosol junctions proved to be mechano-sensitive, suggesting that MKS1 participates in epithelial homeostasis by stabilizing cell junctions, via the transduction of mechanical signals related to epithelial compaction.Functional analysis has shown that the knockdown of MKS1 disrupts the keratin network, and destabilizes the adherent junctions of epithelial cells in culture, with a decrease in the junctional β-catenin and a release of E-cadherin, the α-catenin and vinculin in the cytosol. In addition, the depletion of MKS1 results in a significant decrease in the apical actin network, as well as disorganization of the epithelial structure and a partial transition to a mesenchymal state. These results illustrate a ciliary-independent function of MKS1 in epithelial homeostasis, and provides new hypotheses regarding its role and that of intermediate filaments in epithelial organogenesis processes, in particular tubulogenesis, which is based both on the equilibrium of the epithelium / mesenchyme transition and the mechanotransduction of mechanical stresses during embryogenesisIn order to characterize MKS1 partners, Co-IP experiments and proteomic analyzes have identified epiplakin as a possible MKS1 partner. Epiplakin is a cytolinker capable of binding keratin to membrane and actin; the interaction of MKS1 with epiplakin could thus account for the stabilization of both the keratin network and cell junctions. Additional proteomic analyzes and functional studies will complement these preliminary results.Finally, this work has also revealed the role of MKS1 in the stabilization of gap junctions; the depletion of MKS1 leading to a decrease in the junctional CX43 and an alteration of the intercellular communication function in the epithelial cells in culture. This work, which constitutes the first mention of a possible alteration of gap junctions in this type of disease, will have to be further developed to characterize their impact on tubulogenesis processes.In conclusion, this work which revealed a pre-ciliary role of MKS1 in epithelial homeostasis, provides new hypotheses for the etiology of ciliopathies, previously considered as essentially consecutive to signal transduction defects. It also proposes new mechanisms to account for abnormalities of hepatic development, such as bile ducts dysgenesis, and more broadly tubulogenesis processes involved in the development of many organs.

Einleitung: In der Human- und Veterinärmedizin ist der immunhistologische Keratinnachweis für die Tumordiagnostik von großem Wert. Während beim Menschen bereits spezifische Keratinmuster zur genaueren Charakterisierung epithelialer Neoplasien herangezogen werden können, liegen beim Hund bzgl. der Keratinexpression in Neoplasien nur fragmentarische Kenntnisse vor. Ziele der Untersuchungen: Für die Bewertung der diagnostischen Eignung des Keratinnachweises innerhalb kaniner Neoplasien unter Praxisbedingungen ist eine breit angelegte organübergreifende Betrachtung von Tumoren erforderlich. Ziel dieser Studie war eine umfassende Charakterisierung der Keratinexpression in kaninen epithelialen Neoplasien. Diese erfolgte anhand von Neoplasien unterschiedlicher Histogenese sowie unter Einbezug unterschiedlicher Wachstumsformen dieser Neoplasien sowie ihrer Metastasen. Daneben wurde anhand der in der Humanpathologie bedeutsamen Keratine K7, K8, K13, K14, K19 und K20 auch ein umfangreiches Keratinpanel für die Untersuchung herangezogen. Die Resultate sollten im Kontext der Keratinexpression gesunder kaniner Gewebe betrachtet werden, um die Veränderungen des Expressionsmusters im Rahmen von Neoplasien zu betrachten und deren Eignung für die Charakterisierung von Tumoren zu evaluieren. Tiere, Material und Methoden: Im Rahmen dieser Studie wurden 111 Tumorproben von insgesamt 85 Hunden retrospektiv untersucht. 87 Proben wurden aus 85 primären epithelialen Neoplasien und weitere 24 Proben aus 24 dazugehörigen Metastasen gewonnen. Die Proben wurden anhand der aktuellen WHO-Nomenklatur der histologischen Klassifikation der Tumoren beim Hund in 18 Tumorgruppen und -untergruppen mit, bis auf zwei Ausnahmen, mindestens je 5 Vertretern eingeteilt. Eingang in die Untersuchung fanden dabei Übergangszellkarzinome der Harnblase, Prostatakarzinome, Plattenepithelkarzinome der Haut und Maulschleimhaut, bronchoalveoläre Karzinome der Lunge, Adenokarzinome von Magen, Dünn- und Dickdarm sowie Mammakarzinome mit unterschiedlichen histomorphologischen Erscheinungsbildern. Die routinemäßig aufgearbeiteten Gewebeproben wurden anhand der folgenden kommerziell erhältlichen anti-humanen Anti-Keratin-Antikörper immunhistologisch untersucht: OV-TL 12/30 (Keratin K7), NCL-CK8-TS1 (Keratin K8), AE8 (Keratin K13), NCL-LL002 (Keratin K14), NCL-CK19 (Keratin K19), Ks 20.8 (Keratin K20) und Multikeratinmarker AE1/AE3 (Keratine K1-K8, K10, K13, K14, K15, K16 und K19). Ergebnisse: Insgesamt zeigen die untersuchten Karzinome ein im Vergleich zum gesunden Ursprungsgewebe weitgehend erhaltenes Expressionsmuster, wobei in einigen Fällen jedoch durchaus drastische qualitative und quantitative Abweichungen der Keratinexpression zu beobachten waren. Während in einigen Karzinomen Keratine, welche im orthologen Gewebe nicht nachweisbar waren, beobachtet werden konnten (unerwartete Expression, Neuexpression), trat auch das Fehlen von organtypischen Keratinen innerhalb der Tumoren auf (Expressionsverlust). Obwohl überwiegend eine Konservierung der Keratinexpression innerhalb der Tumoren sichtbar war, fand sich nicht nur zwischen Tumoren unterschiedlicher Organherkunft, sondern auch zwischen Tumoren desselben Organursprungs sowie selbst innerhalb ein und derselben Neoplasie eine mitunter auffällige Variabilität. Trotz dieser inter- und intratumoralen Heterogenität ließen sich Grundmuster der Keratinexpression innerhalb der untersuchten kaninen epithelialen Neoplasien erkennen. So werden die Keratine K7, K8, K13 und K14 in Übergangszellkarzinomen qualitativ und quantitativ variabel exprimiert, während K19 immer und K20 nicht nachweisbar waren. Ein ähnliches Bild zeigt sich für die untersuchten Adenokarzinome, welche jedoch teilweise auch K20 in unterschiedlichem Ausmaß exprimieren. Demgegenüber stehen die Plattenepithelkarzinome der Haut und Maulschleimhaut mit einer typischen K13- und K14-Expression und einem K7- und K20-negativen Phänotyp. Die Expressionsmuster der Metastasen ähneln denen ihrer Primärtumoren. Es konnten jedoch auch hier Abweichungen beobachtet werden. Schlussfolgerungen: Beim Hund bestehen je nach Ursprungsgewebe und Tumorart Unterschiede in der Expression einzelner Keratine. Da für den Hund jedoch neben der teils nicht gegebenen Erhaltung der Keratinmuster eine bisweilen starke Variabilität der Keratinexpression zwischen und innerhalb von Tumoren zu beobachten war und die Keratinmuster der verschiedenen Tumorarten teils breite Übereinstimmungen aufweisen, ist der diagnostische Nutzen der in dieser Studie untersuchten Keratine eingeschränkt. Zwar können Art und Herkunft der Tumoren ausschließlich anhand dieser Auswahl an Keratinmarkern nicht mit absoluter Sicherheit identifiziert werden, allerdings kann der Nachweis einzelner Keratine unter Einbezug von Anamnese, Histomorphologie sowie anderer gewebespezifischer immunhistologischer Marker durchaus eine Diagnose erbringen bzw. eine Verdachtsdiagnose bekräftigen.

The project Kera-Plast aims to re-loop humans and nature by questioning the current systems and ethics through materiality. Human hair, currently considered as waste, functions as the base for the material exploration fabricated through thermo-compression molding. The flexible, short and opaque keratin-fibers get glued together with heat, pressure and water, acting as a plasticizer during the compression molding process. The results are stiff and remind on plastic due to shine and translucency. Aesthetics and function of the resulting material are controlled and designed by traditional textile techniques as knitting, weaving and non-woven processes. The material samples display the potential of Kera-Plast in the categories of 3D surface structures, patterns, shapeability and the influence of light. The findings also provide information about the parameters for designing with keratin fibers through the thermo-compression process. It can be concluded that despite all ethical and cultural factors, Kera-Plast and its fabrication method has the potential to add a sustainable, functional and aesthetical value to the design field and our future material consumption.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21277号 / 農博第2293号 / 新制||農||1062(附属図書館) / 学位論文||H30||N5141(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 杉山 淳司, 教授 吉村 剛, 教授 渡邊 隆司, 教授 高妻 洋成 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Keratins are a family of structural proteins that can be extracted from a variety of sources including wool, nails, skin, hooves, and hair. Keratin can be processed into different constructs such as coatings, scaffolds, and hydrogels, and has shown favorable results when placed in in vitro and in vivo settings for different tissue regeneration applications. Over three decades, keratin extraction technology has been continuously modified, and these differences in extraction processes have distinct effects on the characteristics of the end product. In this work, we examine the effect of keratin aggregation during a widely-used purification step, dialysis ultra-filtration, on material characteristics of the final keratin product when fabricated into a hydrogel. Two distinct dialysis procedures were applied during the extraction of oxidized keratin (keratose): one promoting protein aggregation and the other mitigating it. Analyses of material properties such as mechanical and enzymatic stability were conducted in addition to observing the differences in solution behavior between products. Data revealed that protein aggregation during the extraction process has a profound effect on keratose hydrogel material properties. After determination of the effect of protein aggregation during extraction on keratose hydrogels, investigation of how a blended material comprised of said keratose and type I collagen was undertaken. It was hypothesized that a blend would result in mixing at the molecular level, resulting in improved properties compared to either pure material alone. A protocol was created to make stable keratose/type I collagen blends and material characterization techniques were applied to determine the inherent properties of samples with differing ratios. Crosslinking density, mechanical properties, enzymatic degradation properties, water uptake capacity, structural architecture, and thermal properties were all assessed. In addition, the ability of this material to maintain cell viability was conducted. Results showed that the addition of type I collagen has a significant effect on the properties of hydrogel blends with keratose compared to the pure keratose system. This was mostly evident with hydrogel mechanical stability and material architecture. Finally, the ability to use this hybrid material as a luminal filler for a nerve conduit during peripheral nerve regeneration was explored in an in vitro setting. The ability of this blend to promote Schwann cell viability was assessed in addition to determining the ability of these cells to attach and migrate through the material matrix. These experiments demonstrate proof-of-concept for the application of using keratose/type I collagen matrices as a luminal filler in peripheral nerve guidance conduits. / Doctor of Philosophy / Keratins are a family of structural proteins that can be extracted from wool, skin, nails, and hair, and that have been investigated in the field of tissue regeneration. Humans make several types of keratins, so it has a natural acceptance by the body and its inflammatory and immune systems. However, keratins can be hard to make and process into useful products. Many methods for producing keratin biomaterials have been developed over the past 30 years, but most of them are not ideal. This work sought to explore a production method that addresses a particular problem, that of protein aggregation during purification. In so doing, methods can be optimized to create more useful keratin biomaterials. Experiments comparing preparation methods that maximize and minimize protein aggregation were compared. Data showed that minimizing aggregation leads to better biomaterial characteristics, thus demonstrating the potential impact of targeting this processing step. However, even after optimization of purification, keratins still have limitations. Most notably their mechanical strength is not as great as some other materials. A typical approach to address this in other systems has been by blending. In the present work, we explored a blend made from keratin and type 1 collagen. A method was developed to effectively blend keratin and collagen and create stable mixtures that yielded protein-to-protein coordination. Such interactions typically yield beneficial material characteristics such as increased strength. Data showed that intimate mixing of the two proteins was achieved, and resulting characteristics were improved compared to either pure material. Finally, studies were conducted to assess the potential for keratin/collagen blends to be used to regenerate injured nerves. A common method is to enclose the ends of a cut nerve into a tube and let the nerve re-grow through the tube to its target muscle. An important characteristic is an ability for cells to populate the interior of the tube and help the nerve fibers grow. In the present study, we investigated the behavior of a particularly important cell, the Schwann cell, to attach, move and grow through a keratin/collagen biomaterial. Data showed good cell behavior, suggesting that the material could be used in a medical product for nerve repair.

No / The hair shaft has increasing importance in bioarchaeology, since it is now possible to retrieve detailed biomolecular information on recent life history using individual fibres (e.g., on diet, drug use and DNA). Data on hair condition is an important cornerstone to ensuring that reliable information is obtained. The following study defines morphological features of degradative change in human terminal scalp hair using different microscopy techniques. Evidence of degradative change is translated into a ranked histology for assessing hair sample condition. The approach is applied to samples of cut modern scalp hair subjected to degradation under soil burial/simulated grave conditions.