Glucocorticoids are known to affect metabolic activities of cells. The mechanism of glucocorticoid actions in adult human dermal and mouse L-929 fibroblasts have yet to be fully ascertained. This study endeavors to examine the effects of one glucocorticoid, triamcinolone acetonide, on cellular proliferation and collagen synthesis and to compare such effects in the human and mouse cell lines. Cellular proliferation and collagen synthesis are analyzed and quantitated by cell counts and selective digestion of the protein by bacterial collagenase, respectively. Further analysis of collagen synthesis is provided by polyacrylamide gel electrophoresis. One-tenth triamcinolone acetonide per ml suppresses cellular proliferation of mouse L-929 fibroblasts. Proline incorporation into total and collagenase-sensitive protein is enhanced in the cell layer; that of medium is altered inconsistently. Polyacrylamide gel electrophoresis of proteins treated with pepsin show the abolition of total and collagenase-sensitive protein in the cell layer. Aberrations in hydroxylation and/or deformation in physical structure of protein may confer greater susceptibility to pepsin digestion. Cellular proliferation and proline incorporation into total and collagenase-sensitive protein of adult human dermal fibroblasts are affected inconsistently by the same dose of triamcinolone acetonide. Except for the consistent suppression of cellular proliferation in the murine L-929 fibroblasts by triamcinolone acetonide, all observations pertaining to human dermal fibroblasts are incompatible with those obtained by other workers. Manipulation of culture conditions and glucocorticoid treatment dictate, to a large extent, the kind of responses observed. This could account for the wide variability and frequent contradictory findings reported in the literature. / Pharmaceutical Sciences, Faculty of / Graduate

This study quantified mechanical and structural responses to loading conditions at subtendon hierarchical levels. Tensile tests were performed at three strain rates on three groups of rabbit patellar tendon specimens. For each rate, tangent modulus (E) was computed from the stress-strain curves and the following structural responses were evaluated: (i) Area percent of collagen fibrils (FAR) and (ii) Skewness angle formed between proteoglycans and collagen fibrils. For 0.1%/s, 10%/s, and 70%/s, E was 48.8±20.3MPa, 64.7±29.3MPa, and 78.6±31.7MPa, respectively. For control, 0.1%/s, 10%/s, and 70%/s, the mean FAR was 0.7552±0.1476, 0.6628±0.1190, 0.6335±0.1013, and 0.6047±0.0384, respectively; and proteoglycan skewness angles were 14.70º±11.01º, 12.76º±10.13º, 15.08.0º±11.66º, and 16.68º±12.07º, respectively. For increased E, interfibrillar components had less time for effective fluid flow, energy dissipation, and structural rearrangement. The inverse relationship of FAR to strain rate may be due to broken fibrils and the Poisson effect. Proteoglycan skewness angle increase is likely due to stretched fibrils.

No description available.

Pale, soft, and exudative (PSE) poultry originates during rigor mortis when the muscle pH drops rapidly in high temperature carcasses. This condition results from antemortem stress and/or genetic material in the live animal. PSE poultry is pale in color, has low water-holding capacity, and forms products that are unappealing, dry, and unacceptable to consumers. Since value added products processed with PSE turkey meat display poor protein bind, color, and water retention, enhanced usability could add value to this low value raw material through locating a niche for PSE meat currently utilized in further processed products. Experiment 1 consisted of four broiler breast treatments: 100% PSE, 100% PSE + 1.5% chicken collagen, 100% normal, and 100% normal + 1.5% chicken collagen to test the effects of raw material and chicken collagen. Incorporation of collagen improved (p<0.05) protein bind and CIE L* values in both PSE and normal broiler breast treatments, while decreasing (p<0.05) the cooking and chilling loss of PSE broiler breast treatments. Experiment 2 consisted of four turkey breast treatments: 100% PSE, 100% PSE + 1.5% turkey collagen, 100% normal, and 100% normal + 1.5% turkey collagen to test the effects of raw material and turkey collagen. Addition of turkey collagen improved (p<0.05) the protein bind and CIE L* values in both PSE and normal broiler breast treatments, while decreasing (p<0.05) the cooking and chilling loss of PSE turkey breast treatments. Experiment 3 consisted of five turkey breast treatments: 100% PSE, 100% PSE + 1.5% collagen, 100% PSE + 0.30% kappa/iota carrageenan, 100% PSE + 1.5% soy protein concentrate, and 100% normal to test the effects of raw material, turkey collagen, soy protein concentrate, and carrageenan. Addition of soy protein and turkey collagen both decreased (p<0.05) cooking and chilling loss and increased (p<0.005) the protein bind of 100% PSE. Purge loss was decreased (p<0.05) in PSE raw material when turkey collagen, soy protein concentrate, and kappa/iota carrageenan were utilized. Treatments with collagen displayed similar (p>0.05) CIE L* and CIE a* values to that of normal treatments. No differences (p>0.05) in consumer acceptability existed among the treatments. / Master of Science

It is well documented that the incidence of renal disease, and therefore renal dysfunction, increases with age in many species of mammals. Such alterations in renal structure and function may significantly affect long-term toxicology studies. The purpose of this study was to assess the temporal evolution of glomerulosclerosis, an important renal lesion, in laboratory housed dogs, an important model system in chronic toxicological studies. We histopathologically examined representative sections of dog kidneys, quantified glomerular lesions (using the 0-5 scale of the World Health Organization classification system) and performed of statistical analysis of the extent and distribution of such changes. The kidney samples were obtained by necropsy, and occasionally biopsy, procedures from a collection of 159 purebred Beagle dogs maintained for their entire lifespan in well-controlled conditions. The lesions were correlated with sex, age, and intra-renal location of affected glomeruli to determine the relationship of each in the development of glomerulosclerosis. All dogs examined had some degree of glomerulosclerosis. In the youngest (up to 2 years of age), this was minimal, but was more advanced by middle age (3-7 years). The condition progressed with further aging and was associated with progressive fibrosis and tubular loss. Location and advancing age were significantly related to the development of glomerulosclerosis such that as age increases, the incidence of glomerulosclerosis increases, with the inner medullary ray and inner cortex demonstrating the highest occurrence. Using immunohistochemical analysis, the percentage of type IV collagen within glomeruli was determined. No significant increase in type IV collagen in glomeruli due to age or location was seen. An increase in type III or type V collagen within glomeruli was not apparent either, upon visual examination. This study indicates that renal lesions, including glomerulosclerosis, occur commonly and progress over the lifetime in a genetically similar population of laboratory Beagle dogs maintained under optimal standard environmental conditions. Such typical, age-related change needs to be taken into consideration when conducting chronic toxicological experiments using such animals. / Master of Science

No

Collagen is one of the major protein constituents in mammals and is present in all tissues and organs with the exceptions of keratin tissues such as hair and nails. Collagen monomers self-aggregate into a number of structures. In order to understand the physical bases for the structural polymorphism observed in collagen, a good starting point is one of the simplest collagen aggregates, segmental long spacing (SLS) collagen. Although SLS collagen formation induced by the presence of adenosine 5’-triphosphate is widely known, effects of other triphosphates, on the other hand, are much less studied. By varying the pH, it is discovered that all the nucleoside 5’-triphophsates, as well as inorganic triphosphate, are able to induce SLS formation over certain pH ranges. Adenosine 5’-diphosphate and para-nitrophenylphosphate cannot induce SLS formation at any pH. Based on the pH ranges at which SLS collagen can be formed, it is concluded the triphosphate functionality, with one negative charge per phosphate group, is primarily responsible for the formation of SLS collagen. Since inorganic triphosphate is able to induce SLS collagen formation, the presence of the nucleoside is optional for the assembly process; however if present, the assembly process prefers the nucleosides carrying acidic protons. Using electron spectroscopic imaging (ESI) technique, it is found phosphorus, present only in nucleotides but not in polypeptides, is localized in certain regions of SLS collagen, forming a unique banding pattern transverse the long axis of the SLS collagen. Nitrogen mapping indicates the localization of phosphorus is not due to accumulation of materials. The phosphorus banding pattern demonstrates an excellent consistency across SLS collagen assembled from both bovine and recombinant human collagen monomers. Results from molecular simulation are consistent with the experimental results. All threephosphate groups seem to be involved in the assembly process to some degree. In the last chapter of the thesis, a reliable protocol to synthesis native type collagen fibers is introduced.

Collagen is one of the major protein constituents in mammals and is present in all tissues and organs with the exceptions of keratin tissues such as hair and nails. Collagen monomers self-aggregate into a number of structures. In order to understand the physical bases for the structural polymorphism observed in collagen, a good starting point is one of the simplest collagen aggregates, segmental long spacing (SLS) collagen. Although SLS collagen formation induced by the presence of adenosine 5’-triphosphate is widely known, effects of other triphosphates, on the other hand, are much less studied. By varying the pH, it is discovered that all the nucleoside 5’-triphophsates, as well as inorganic triphosphate, are able to induce SLS formation over certain pH ranges. Adenosine 5’-diphosphate and para-nitrophenylphosphate cannot induce SLS formation at any pH. Based on the pH ranges at which SLS collagen can be formed, it is concluded the triphosphate functionality, with one negative charge per phosphate group, is primarily responsible for the formation of SLS collagen. Since inorganic triphosphate is able to induce SLS collagen formation, the presence of the nucleoside is optional for the assembly process; however if present, the assembly process prefers the nucleosides carrying acidic protons. Using electron spectroscopic imaging (ESI) technique, it is found phosphorus, present only in nucleotides but not in polypeptides, is localized in certain regions of SLS collagen, forming a unique banding pattern transverse the long axis of the SLS collagen. Nitrogen mapping indicates the localization of phosphorus is not due to accumulation of materials. The phosphorus banding pattern demonstrates an excellent consistency across SLS collagen assembled from both bovine and recombinant human collagen monomers. Results from molecular simulation are consistent with the experimental results. All threephosphate groups seem to be involved in the assembly process to some degree. In the last chapter of the thesis, a reliable protocol to synthesis native type collagen fibers is introduced.

Sampling methods for dentine and bone collagen have been evolving for several decades. Incremental dentine collagen sampling and bone collagen sampling have been limited by the available technology throughout that time. As the technology for isotope ratio mass spectrometry analysis improves, the sampling methods should improve as well. This research focused on developing a new incremental dentine collagen sampling method and bone collagen microsampling method for stable isotope analysis. This research aimed to increase the temporal resolution of incremental dentine collagen sampling and provided sequential collagen sampling from bone collagen for stable carbon and nitrogen isotope analysis while limiting the destructive nature of bioarchaeological analysis. It was determined that the temporal resolution for incremental analysis could be reduced to approximate three months, opposed to the nine months found in other sampling methods. It was also determined that detailed isotopic data could be obtained from bone collagen when sampling the microstructures. The increased amount of isotopic data from the bone collagen was an improvement on the commonly used bulk collagen sampling. This research can be utilised to answer several of the questions that archaeologists have been asking about past populations. Isotopic analysis using the methods developed in the research can provide a more detailed observation of the diet and health of past populations. In addition, the developed methods for bone and dentine collagen reduced the amount of tissue subjected to destructive analysis.

Collagen is the most abundant protein in mammals and has special mechanical behavior that enables it to play an important role in the structural integrity of many tissues, e.g., skin, tendon, bone, cartilage and blood vessels. The mechanical properties of collagen are governed by hierarchical mechanisms in different length-scales from molecule to tissue level. Currently, there is no multi-scale model that can predict the mechanical properties of collagen at macroscopic length scales from the behavior of microstructural elements at smaller length scales. This dissertation aimed at developing a multi-scale model using a bottom-up approach to predict the elastic and viscoelastic behaviors of collagen at length scales spanning from nano to microscale. Creep simulations were performed using steered molecular dynamics (SMD) method on collagen molecules, cross-link, and micro-fibrils with various lengths. A micro-fibril is considered as a combination of two collagen molecules connected by a cross-link. The strain time histories for force levels in the range of 10 to 4000 pN were characterized using quasilinear viscoelastic models. These models were utilized to make a reduced model of a micro-fibril and the reduced models, in turn, were combined to make a model of a fibril up to 300 micrometers in length. The micro-fibril and fibril models were validated with available experimental measurements. Hydrogen bonds rupture and formation of collagen molecule played a central role in its viscoelastic behavior and were used to estimate the creep growth rate. The propagation of force wave in the molecule was shown to be an important factor in providing the time-dependent properties of the fibrils. This propagation was modeled with delay elements and this allowed reducing the micro-fibril model to only three degrees of freedom. In conclusion, the results confirmed that the combination of molecular dynamics simulations and viscoelastic theory could be successfully utilized to investigate the viscoelastic behavior of collagen at small scales. The model reported in this dissertation, lays the groundwork for future studies on collagen, particularly in elucidating how each particular level of hierarchy affects the overall tissue behavior. / Mechanical Engineering