No description available.

This thesis describes the study of the molecular packing and organisation of collagen molecules within a fibril. The first two chapters describe the background to the study. In Chapter 1, a review of the extracellular matrix concentrates on the structure and organisation of type I collagen. Chapter 2 summarises the theory of X-ray diffraction by fibres, and Chapter 3 describes X-ray sources and equipment used in data collection. Data treatments and data extraction methods (such as simulated annealing) are also discussed. Chapters4 and 5 present the results of the study. Chapter 4 describes the determination of the one-dimensional structure of type I collagen to 0.54 nm resolution using X-ray diffraction and isomorphous derivative phase determination. The significance of the electron density map is interpreted in light of the known amino acid sequence, showing possible variations in the nature of the helix pitch. More importantly, the conformations of the intermolecular crosslink forming non-helical telopeptides were determined. Chapter 5 provides a detailed background to the current understanding of the three dimensional packing structure of collagen, and presents the first model-independent phase determined structure of a natural fibre - the lateral packing structure of type I collagen in rat tail tendon. The data extraction methods described in Chapter 3 are employed to calculate an electron density map of anisotropic resolution, from which the 4 crosslink forming telopeptide segments within the quasi-hexagonal packing structure are identified. Conclusions are drawn concerning the nature of order/disorder within collagen fibrils and the validity of the compressed microfibril model of collagen molecular packing and organisation is discussed. Chapter 6 summaries the results and evaluates the success of the study. The potential for development of the techniques and results found for further studies are also discussed.

Content: In leather making processes, the thermal and non-thermal effect of microwave, especially non-thermal effect, strengthen the combination between collagen and chemicals. Although tanning under microwave makes the leather have better thermal stability, the tanning process and leather properties have not been studied in detail. For illustrating the influence of microwave on chrome tanning process, pickled skin was tanned for 6h as penetration procedure and then basified for another 4h as fixation procedure. The tanning under microwave heating (MW) was experimental sample and under water bath heating was control. UVVis, ICP-OES and pH meter were used to measure the changes of tanning effluent during tanning, and Shrinkage temperature meter, DSC, TG, FT-IR, SEM, XRD and XPS were applied to determine the differences between MW and WB in aspect of leather property and structure. The results indicated microwave accelerated chrome tanning agent penetration and had better promotion effect on chromium complex hydrolysis and olation. The leather tanned with microwave assisting had special effect on improve tanning effect which led better thermal stability and resistance of leather, but the collagen structure, including triple helix structure, stayed as WB and the combination mechanism between collagen and chromium was also same with conventional. In sum, microwave had positive effect on accelerating tanning rate and resulting in better leather without any negative effect on leather structure. Therefore, microwave would be a potential for achieving clean and sustainable chrome tanning by making tanning much faster and more efficiency. Take-Away: Microwave promotes chrome tanning agent penetration and combination Microwave has positive effect on tanning effect further as the leather tanned by microwave assisting has higher thermal stability. Although microwave promotes chrome tanning process, the collagen structure and tanning mechanism remained as normal.

Content: Pre-treatments are widely used during tanning processes as to improve the performance of the main tannage. Synchrotron small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) were used to study four common types of pre-treatments, viz. monodentate complexing agent (sodium formate, SF), chelating agent (disodium phthalate, DSP), covalent cross-linker (glutaraldehyde, GA) and nanoclay (sodium montmorillonite, MMT) about their effects on chromium-collagen cross-linking reaction during tanning. Based on the results, the performance of chromium-collagen cross-linking with and without pre-treatments was presented considering five aspects: cross-linking, the level of hydration, hydrothermal stability, uniformity through leather cross-section and the uptake of chrome. Comparing to the original ThruBlu chrome tanning, at the same chrome offers, leather pre-treated using SF, DSP and MMT showed improved hydrothermal stability, uniformity and the level of hydration, while GA showed decreased hydration. All of the pre-treatments reduce surface fixation by decreasing the reactivity of chromium with collagen. Changes in the reaction performance can influence the properties of the leather products as well as the efficiency of the leather manufacturing processes. Insights into the structural changes of collagen during tanning with varied reaction conditions can guide the design of novel, benign tanning processes to reduce environmental impact. Take-Away: 1. Uniformity of the hydrothermal stability through leather cross-section were improved by all of the studied pre-treatments. 2. Reactivity of chromium to cross-link with collagen was reduced as a result of the complexing, covalent cross-linking, or preferential adsorption. 3. Complexing agents and nanoclay pre-treatments tend to retain collagen bound water, while covalent cross-linker causing decrease in the level of hydration of collagen.

Cell fate is known to be triggered by cues from the extracellular matrix including its chemical, biological and physical characteristics. Specifically, mechanical and topological properties are increasingly recognized as important signals. The aim of this work was to provide an easy-accessible biomimetic in vitro platform of topologically defined collagen I matrices to dissect cell behaviour under various conditions in vitro. We reconstituted covalently bound layers of three-dimensional (3D) networks of collagen type I and collagen type V with a defined network topology. A new erosion algorithm enabled us to analyse the mean pore diameter and fibril content, while the mean fibril diameter was examined by an autocorrelation method. Different concentrations and ratios of collagen I and V resulted in pore diameters from 2.4 μm to 4.5 μm and fibril diameters from 0.6 to 0.8 μm. A comparison of telopeptide intact collagen I to telopeptide deficient collagen I revealed obvious differences in network structure. The good correlation of the topological data to measurements of network stiffness as well as invasion of human dermal fibroblasts proofed the topological analysis to provide meaningful measures of the functional characteristics of the reconstituted 3D collagen matrices.

<p>In 2011, an estimated 230,480 new cases of invasive breast cancer were diagnosed among women, as well as an estimated 57,650 additional cases of in situ breast cancer [1]. Breast conserving surgery (BCS) is a recommended surgical choice for women with early stage breast cancer (stages 0, I, II) and for those with Stage II-III disease who undergo successful neo-adjuvant treatment to reduce their tumor burden [2, 3]. Cancer within 2mm of a margin following BCS increases the risk of local recurrence and mortality [4-6]. Margin assessment presents an unmet clinical need. Breast tissue is markedly heterogeneous which makes identifying cancer foci within benign tissue challenging. Optical spectroscopy can provide surgeons with intra-operative diagnostic tools. Here, ex-vivo breast tissue is evaluated to determine which sources of optical contrast have the potential to detect malignancy at the margins in women of differing breast composition. Then, H&E images of ex-vivo breast tissue sites are quantified to further deconstruct the relationship between optical scattering and the underlying tissue morphology. </p><p>Diffuse reflectance spectra were measured from benign and malignant sites from the margins of lumpectomy specimens. Benign and malignant sites were compared and then stratified by tissue type and depth. The median and median absolute deviance (MAD) was calculated for each category. The frequencies of the benign tissue types were separated by menopausal status and compared to the corresponding optical properties. </p><p>H&E images were then taken of the malignant and benign sites and quantified to describe the % adipose, % collagen and % glands. Adipose sites, images at 10x, were predominantly fatty and quantified according to adipocyte morphology. H&E-stained adipose tissue sections were analyzed with an automated image processing algorithm to extract average cell area and cell density. Non-adipose sites were imaged with a 2.5x objective. Grids of 200µm boxes corresponding to the 3mm x 2mm area were overlaid on each non-adipose image. The non-adipose images were classified as the following: adipose and collagen (fibroadipose); collagen and glands (fibroglandular); adipose, collagen and glands (mixed); and malignant sites. Correlations between <&mus&#8242;> and % collagen in were determined in benign sites. Age, BMI, and MBD were then correlated to <&mus&#8242;> in the adipose and non-adipose sites. Variability in <&mus&#8242;> was determined to be related to collagen and not adipose content. In order to further investigate this relationship, the importance of age, BMI and MBD was analyzed after adjusting for the % collagen. Lastly, the relationship between % collagen and % glands was analyzed to determine the relative contributions of % collagen and % glands <&mus&#8242;>. Statistics were calculated using Wilcoxon rank-sum tests, Pearson correlation coefficients and linear fits in R. </p><p> The diagnostic ability of the optical parameters was linked to the distance of tumor from the margin as well as menopausal status. [THb] showed statistical differences from <&mus&#8242;> between malignant (<&mus&#8242;>: 8.96cm-1±2.24MAD, [THb]: 42.70&muM±29.31MAD) compared to benign sites (<&mus&#8242;>: 7.29cm-1±2.15MAD, [THb]: 32.09&muM±16.73MAD) (p<0.05). Fibroglandular (FG) sites exhibited increased <&mus&#8242;> while adipose sites showed increased [&beta-carotene] within benign tissues. Scattering differentiated between ductal carcinoma in situ (DCIS) (9.46cm-1±1.06MAD) and invasive ductal carcinoma (IDC) (8.00cm-1±1.81MAD), versus adipose sites (6.50cm-1±1.95MAD). [&beta-carotene] showed marginal differences between DCIS (19.00&muM±6.93MAD, and FG (15.30&muM±5.64MAD). [THb] exhibited statistical differences between positive sites (92.57&muM±18.46MAD) and FG (34.12&muM±22.77MAD), FA (28.63&muM±14.19MAD), and A (30.36&muM±14.86MAD). Due to decreased fibrous content and increased adipose content, benign sites in post-menopausal patients exhibited lower <&mus&#8242;>, but higher [&beta-carotene] than pre-menopausal patients.</p><p>Further deconstructing the relationship between optical scattering and tissue morphology resulted in a positive relationship between <&mus&#8242;> and % collagen (r=0.28, p=0.00034). Increased variability was observed in sites with a higher percentage of collagen. In adipose tissues MBD was negatively correlated with age (r=-0.19, p=0.006), BMI (r=-0.33, p=2.3e-6) and average cell area (r=-0.15, p=0.032) but positively related to the log of the average cell density (r=0.17, p=0.12). In addition, BMI was positively correlated to average cell area (r=0.31, p=1.2e-5) and negatively related to log of the cell density (r=-0.28, p=7.6e-5). In non-adipose sites, age was negatively correlated to <&mus&#8242;> in benign (r=-0.32, p=4.7e-5) and malignant (r=-0.32, p=1.4e-5) sites and this correlation varied significantly by the collagen level (r=-0.40 vs. -0.13). BMI was negatively correlated to <&mus&#8242;> in benign (r=-0.32, p=4e-5) and malignant (r=-0.31, p=2.8e-5) sites but this relationship did not vary by collagen level. MBD was positively correlated to <&mus&#8242;> in benign (r=0.22, p=0.01) and malignant (r=0.21, p=4.6e-3) sites. Optical scattering was shown to be tied to patient demographics. Lastly, the analysis of collagen vs. glands was narrowed to investigate sites with glands between 0-40% (the dynamic range of the data), the linear model reflected an equivalent relationship to scattering from % glands and the % collagen in benign sites (r=0.18 vs. r=0.17). In addition, the malignant sites showed a stronger positive relationship (r=0.64, p=0.005) to <&mus&#8242;> compared to the benign sites (r=0.52, p=0.03).</p><p>The data indicate that the ability of an optical parameter to differentiate benign from malignant breast tissues is dictated by patient demographics. Scattering differentiated between malignant and adipose sites and would be most effective in post-menopausal women. [&beta-carotene] or [THb] may be more applicable in pre-menopausal women to differentiate malignant from fibrous sites. Patient demographics are therefore an important component to incorporate into optical characterization of breast specimens. Through the subsequent stepwise analysis of tissue morphology, <&mus&#8242;> was positively correlated to collagen and negatively correlated to age and BMI. Increased variability of <&mus&#8242;> with collagen level was not dependent on the adipose contribution. A stronger correlation between age and <&mus&#8242;> was seen in high collagen sites compared to low collagen sites. Contributions from collagen and glands to <&mus&#8242;> were independent and equivalent in benign sites; glands showed a stronger correlation to <&mus&#8242;> in malignant sites than collagen. This information will help develop improved scattering models and additional technologies from separating fibroglandular sites from malignant sites and ultimately improve margin assessment.</p> / Dissertation

Content: A comprehensive analysis of the molecular and structural components of deer skin and cow hide was undertaken. These skins known to be strong, however they derive their strength from different combinations of molecular and structural properties. Firstly, the physical properties of deer skin and cow hide including the tensile strength, tear strength and denaturation temperature were measured. Secondly, the structure of the collagen fibrils and glycosaminoglycans was investigated using transmission electron microscopy (TEM), and small angle X-ray scattering (SAXS). Finally, the chemical composition of deer skin and cow hide such as amino acids, crosslinks and glycosaminoglycans were analysed. Our results showed that physical properties of deer skin and cow hide are derived from different combinations of several chemical components resulting in different architecture. It was found that the large and “wavy” collagen fibres in deer skin made up of collagen fibrils with small diameters. Additionally, deer skin fibrils appeared to be linked by regular arrays of filaments of large glycosaminoglycans that are distributed uniformly. Deer skin contained higher proportion of trivalent collagen crosslinks. In contrast, the collagen fibrils in cow hide were large, contained a diverse glycosaminoglycan distribution and a higher proportion of tetravalent collagen crosslinks, resulting in straight collagen fibres. This study suggests that although deer skin and cow hide are both strong, they have different structural and molecular features. Take-Away: Deer skin and cow hide have different structural and molecular make up which are reflected in their physical properties particularly strength. Glycosaminoglycans are important for the organisation of collagen fibrils in deer skin and cow hide. Deer skin and cow hide contain different ratios of collagen natural crosslinks which are essential collagen stability.

Content: Synchrotron small-angle X-ray scattering (SAXS) technique was applied to leather research to understand the changes in molecular-level collagen structure during tanning and denaturation (shrinkage), which can help reduce the environmental impact from the extensive chrome usage. Based on SAXS results from realtime denaturation experiments on leather samples, we established a mechanistic model of chrome tanning indicated by the structural changes of collagen. It suggests that only a low level of chromium species is effectively involved in the cross-linking with collagen, which highlights the overuse of chrome during conventional tanning processes. Any extra amount of chrome added, however, can support the stabilisation of collagen possibly via a non-covalent mechanism. Such mechanism points towards a more environment-friendly tanning method by using suitable supplementary reagents to benefit tanning effect non-covalently instead of chrome. By pre-treating with complexing agents such as sodium formate and disodium phthalate, as well as nanoclay (sodium montmorillonite), the uniformity through bovine hide collagen matrix were improved significantly. These pre-treatments effectively reduce the reactivity of chromium during its cross-linking reaction with collagen while retaining its bound water. However, collagen pre-treated with a covalent cross-linker (glutaraldehyde) results in a decrease in both chromium-collagen cross-linking and bound water while improving uniformity. These molecular-level insights can be developed into metrics to guide us towards a more sustainable future for the leather industry. The coating on collagen fibrils can provide a pseudo-stabilisation effect of increasing the heat resistance of collagen. A group of tanning experiments were also conducted in situ on the SAXS beamline to observe the different mechanisms of free-of-chrome alternative tannages as potential replacements. Overall, synchrotron SAXS provides valuable information about collagen structure changes that could lead to more efficient use of chrome (or other tanning agents) in the global leather tanning industry. Take-Away: 1. Kinetics of hydrothermal denaturation and collagen crosslinking mechanism proceed through different pathways 2. Alternative chemistries to enhance chrome uptake at low offers. 3. The role of collagen structure studies in understanding tanning mechanims