Investigations of the physical and chemical structure of archaeological fibresArchaeological fibres can be defined as natural fibres that belong to different time periods, which found in cemeteries or excavation sites. The preservation conditions cause degradation, mineralisation and sometimes a complete deterioration of these fibres, because the chemical and physical structure of the fibres changed over time in response to the specific burial environments. The ancient fibres from different archaeological sites were analysed by several non destructive analytical techniques such as optical Microscopy, Environmental Scanning Electron Microscopy, Attenuated Total Reflectance FTIR and Wide Angle X-Ray Scattering Analysis as well as destructive analytical techniques such as Scanning Electron Microscopy, Transmission Fourier Transform Infrared Spectroscopy, Energy Dispersive X-Ray Spectroscopy and Differential Scanning Calorimetry. These analytical techniques showed that keratin fibres from a central European climate have a larger damage at the fibre surface compared with frozen conditions. While bog conditions were the best in preserving the surface. FTIR analysis provides information about cystine oxidation changes in keratin fibres. For all ancient keratin fibres showed a silica peak at 1030 cm-1 which affected the symmetric cysteic acid peak at 1040 cm-1. For this reason the asymmetrical cysteic acid peak 1175 cm-1 was used for identification of cystine oxidation changes. Transmission FTIR gives a better view of the overall chemical changes in both cortex and cuticle compared to ATR analysis. All ancient wools and highly medullated Iceman deer hairs showed the highest concentration of cysteic acid compared with human hair and goat hair. Also it was shown that warm conditions have bigger effect on both the degree of oxidation of cystine and the ions uptake from the environment. The modulated DSC analysis gives a better view on the degree of degradation of hair proteins compared to WAXS analysis. To get a reliable result it is important to correct the DSC data according to the protein content of the fibre.

Restriction enzyme, molecular cloning and DNA annealing techniques have been used to study mRNA and DNA coding for the embryonic feather keratins of the chicken and the DNA genomes of coliphages 186 and P2. The coliphage DNAs were used to develop the techniques for application to the keratin system which awaited the availability of appropriate bio - hazard containment facilities before being undertaken. The following results were obtained. 1. Restriction endonuclease cleavage of chick DNA with BamHI, BgïII, EcoRI, or HindIII, fractionation on agarose gels, immobilization on nitrocellulose filters and annealing to DNA complementary to purified 12S mRNA isolated from the developing embryonic feather and coding for embryonic feather keratins, yielded a complex pattern of major and minor bands. These patterns consisted of 4 - 6 major bands and many minor bands. No simple repeat length could be deduced from these patterns, suggesting that keratin - coding DNA is heterogeneous in coding sequences, non - coding sequences or both. 2. Keratin gene expression was shown to be independent of DNA rearrangement, as the complex pattern of restriction fragments was identical in DNA isolated from germ - line tissue ( sperm ) the differentiated feather tissue and somatic tissue not synthesizing keratins ( erythrocytes ). Keratin gene expression must therefore involve the activation of pre - existing control regions in the DNA. 3. The purified 12S mRNA coding for feather keratin was transcribed into double - stranded DNA and individual species isolated by molecular cloning in E. coli. Sequence variation between species was confirmed by restriction enzyme analysis. 4. Preliminary analysis of the cloned species revealed the existence of two distinct groups of species comprising 12S mRNA : Group I ( the more abundant group ) and Group II ( the less abundant ). The fact that filter - bound DNA of individual Group I species bound more 12s cDNA than equal amounts of Group II species DNA and that pure Group I species and total 12S mRNA sequences ( coding for keratins in cell - free translation systems ) annealed to exactly the same complex set of EcoRI, HindIII, or BgïII restricted chick DNA fragments, compels the conclusion that Group I species represent true keratin coding sequences. Group II species annealed to restricted chick DNA fragments which were totally different to those annealing, to either Group I species or total 12S mRNA sequences. Different Group II species appeared to anneal to certain common fragments, suggesting that this less abundant group was comprised of a family of sequence related species and were not simply contaminating mRNA species coding for ' housekeeping ' functions. Their exact nature is at present, however, uncertain. 5. Group I species, the presumptive keratin - coding species, are members of a family of homologous species present in the chick genome. This is demonstrated by the fact that the two Group I species which have been examined so far, shown to be non - identical by restriction analysis, and total 12S mRNA sequences from which they were derived, annealed to the same set of between 20 and 30 BglII, HindIII or EcoRI restricted chick DNA fragments under annealing and washing conditions of low stringency, ( high salt ). Under stringent ( low salt ) washing conditions, however, all except between 1 and 3 of the duplexes formed by these fragments and the Group I species were differentially lost from the filter, indicating that the majority of duplexes were mis - matched and therefore that these multiple copies were homologous and not identical. In addition the two non - identical Group I species annealed to EcoRI generated chick DNA fragments of different sizes under the stringent ( low salt ) washing conditions, demonstrating that differences must exist in the sequence of adjacent non - coding and / or intervening sequences ( should they exist ) for these two species. 6. Although the two Group I species discussed above annealed to different EcoRI generated chick DNA fragments under the stringent ( low salt ) washing conditions, they both annealed under these conditions to a HindIII generated chick DNA fragment of size 3.0 kb. Assuming that this is a single fragment and not two fragments co - electrophoresing by chance, sequences identical to or with very close homology to both of these species lie on the same fragment and are therefore linked in the genome. The exact nature of this linkage and of the extent of gene clustering, should it exist, was not determined. 7. Restriction cleavage maps of coliphages 186 and P2 were determined for the enzymes BamHI, BglII, EcoRI, HindIII, PstI, SaïI, XbaI, and XhoI. These maps were used to analyse four insertion or deletion mutants affecting the major control region of 186. 186ins2 and 186ins3 were shown to be insertions of an IS3 element in the cI. gene and int gene respectively. 186dell and 186del2 were shown to carry the same deletion affecting the cI gene, but 186del2 carried a cryptic insert in the repressor binding site ( operator ). / Thesis (Ph.D.)--Department of Biochemistry, 1979.

Bibliography: leaves 87-113. / xi, 113, [72] leaves, [42] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Presents results from an investigation into the regulation of hair-specific gene expression, including attempts to produce an immortalised hair follicle cortical cell line for this purpose and the use of mouse transgenesis and invitro gel mobility shift assays. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1995

Thesis (Ph.D. 1979) from the Department of Biochemistry, University of Adelaide.

Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 255).

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The inherent design freedom of protein engineering and recombinant protein production enables specific tailoring of protein structure, function, and properties. Two areas of research where protein engineering has allowed for many advances in biomedical materials include the design of novel protein scaffolds for molecular recognition, as well as the use of recombinant proteins for production of next generation biomaterials. The main focus of my dissertation was to develop new biomedical materials using protein engineering. Chapters three and four discuss the engineering of repeat proteins as bio-recognition modules for biomedical sensing and imaging. Chapter three provides an overview of the most recent advances in engineering of repeat proteins in the aforementioned field. Chapter four discusses my contribution to this field. We have designed a de novo repeat protein scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Innate immunity receptors have been described as pattern recognition receptors in that they recognize "global features" of a family of pathogens versus one specific antigen. In mammals, two main protein families of such receptors are: extracellular Toll-like receptors (TLRs) and cytoplasmic Nucletide-binding domain- and Leucine-rich Repeat-containing proteins (NLRs). NLRs are defined by their tripartite domain architecture that contains a C-terminal LRR (Leucine Rich Repeat) domain, the nucleotide-binding oligomerization (NACHT) domain, and the N-terminal effector domain. It is proposed that pathogen sensing in NLRs occurs through ligand binding by the LRR domain. Thus, we hypothesized that LRRs would be suitable for the design of alternative binding scaffolds for use in molecular recognition. The NOD protein family plays a very important role in innate immunity, and consequently serves as a promising scaffold for design of novel recognition motifs. However, engineering of de novo proteins based on the NOD family LRR domain has proven challenging due to problems arising from protein solubility and stability. Consensus sequence design is a protein design tool used to create novel proteins that capture sequence-structure relationships and interactions present in nature in order to create a stable protein scaffold. We implement a consensus sequence design approach to develop proteins based on the LRR domain of NLRs. Using a multiple sequence alignment we analyzed all individual LRRs found in mammalian NLRs. This design resulted in a consensus sequence protein containing two internal repeats and separate N- and C- capping repeats named CLRR2. Using biophysical characterization methods of size exclusion chromatography, circular dichroism, and fluorescence, CLRR2 was found to be a stable, monomeric, and cysteine free scaffold. Additionally, CLRR2, without any affinity maturation, displayed micromolar binding affinity for muramyl dipeptide (MDP), a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand. Furthermore, CLRR2 demonstrated selective recognition to the biologically active stereoisomer of MDP. Results of this study indicate that LRRs are indeed a useful scaffold for development of specific and selective proteins for molecular recognition, creating much potential for future engineering of alternative protein scaffolds for biomedical applications. My second research interest focused on the development of proteins for novel biomaterials. In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues, as well as participates in important regulatory functions such as cell migration and proliferation and protein signalling. The aforementioned properties along with keratins' inherent capacity for self-assembly poise it as a promising scaffold for regenerative medicine and tissue engineering applications. However, due to the extraction process used to obtain natural keratin proteins from natural sources, protein damage and formation of by-products that alter network self-assembly and bioactivity often occur as a result of the extensive processing conditions required. Furthermore, natural keratins require exogenous chemistry in order to modify their properties, which greatly limits sequence tunability. Recombinant keratin proteins have the potential to overcome the limitations associated with the use of natural keratins while also maintaining their desired structural and chemical characteristics. Thus, we have used recombinant DNA technology for the production of human hair keratins, keratin 31 (K31) and keratin 81 (K81). The production of recombinant human hair keratins resulted in isolated proteins of the correct sequence and molecular weight determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry. Proteins with no unwanted sequence truncations, deletions, or mutations indicate recombinant DNA technology can be used to reliably generate full length keratin proteins. This allows for consistent starting materials with no observable impurities or undesired by-products, which combats a major challenge associated with natural keratins. Additionally, recombinant keratins must maintain the intrinsic propensity for self-assembly found in natural keratins. To test the propensity for self-assembly, we implemented size exclusion chromatography (SEC), dynamic light scattering (DLS), and transmission electron microscopy (TEM) to characterize K31, K81, and an equimolar mixture of K31 and K81. The results of the recombinant protein characterization reveal novel homo-polymerization of K31 and K81, not previously reported, and formation of characteristic keratin fibers for the K31 and K81 mixture. Therefore, recombinant K31 and K81 retain the intrinsic biological activity (i.e. self-assembly) of natural keratin proteins. We have also conducted a comparative study of recombinant and extracted heteropolymer K31/K81. Through solution characterization and TEM analysis it was found that use of the recombinant heteropolymer allows for increased purity of starting material while also maintaining self-assembly properties necessary for functional use in biomaterials design. However, under the processing condition implemented, extracted keratins demonstrated increased efficiency of assembly. Through each study we conclude that recombinant keratin proteins provide a promising solution to overcome the challenges associated with natural protein materials and present an exceptional design platform for generation of new biomaterials for regenerative medicine and tissue engineering. / Ph. D.

Keratin is a biomaterial derived from biological sources and can be used in a variety of medical applications. This study focuses on keratin derived from human hair. Unfortunately, there is not a lot of information in the literature describing how keratin reacts to subtle changes in an aqueous solution such as differences in pH, keratin concentration, buffer concentration, salt concentration, and temperature. To have a better understanding of this effect, dynamic light scattering was used to test the size ranges and volume percentages in each range. Dynamic light scattering shows the size of the keratin in each environment and its consistency with time. The results showed that there is a difference in keratin behavior between water and buffer solutions, but very subtle differences between each buffer, buffer concentration, keratin concentration, pH and temperature. Keratins aggregate extensively in un-buffered conditions (i.e. pure water), which has implications to both purification and fabrication of biomaterials as water is used extensively in these processes. Interestingly, there was little effect of keratin concentration, pH, and temperature on the buffers used in this study, suggesting there may be a wide range of conditions in which aggregation can be minimized. / Master of Science

Keratins are fibrous structural proteins found in human hair that have been used to develop bioactive and biocompatible constructs for a wide variety of tissue engineering and healthcare applications. Their ubiquity, capacity for self-assembly, ease of use and versatility in blended materials, and ability to modulate cell behavior and promote tissue ingrowth have made keratins well-suited for the development of regenerative therapies. In particular, keratins have demonstrated bioactivity in both in-vivo and in-vitro studies, by altering immune and stem cell phenotype and function and promoting an anti-inflammatory/wound healing environment. This work seeks to build on previous research by investigating the ability of low and high molecular weight keratins to augment anti-inflammatory primary macrophage phenotypes and examining the influence of keratin biomaterials on cellular and inflammatory mechanisms in two models of injury and disease. Rodent models of blast induced neurotrauma (BINT) and severe osteoporosis were used to inform the development of 2D and 3D in-vitro models of macrophage/endothelial cell injury and osteogenic differentiation respectively. Keratin biomaterials exhibited some potential to alter macrophage and endothelial cell dynamics following blast, specifically by promoting anti-inflammatory (M2c-like) macrophage polarization and diminishing endothelial cell injury responses (i.e. endothelial glycocalyx shedding). A more clinically relevant model of osteoporosis found that stem cells harvested from older, osteoporotic animals demonstrated limited proliferative and bone differentiation potential compared to healthy cells. However, 3D constructs (especially keratin-based materials) were able to enhance calcification and osteogenic gene expression of diseased cells. These results highlight the complexity of macrophage phenotypic switching and cellular dynamics in these systems. However, keratin-based therapeutics may prove useful for facilitating tissue regeneration and limiting detrimental inflammatory and cellular responses in various models of injury and disease. / Ph. D.