Thesis (M.S.)--University of Michigan, 1991.

Thesis (Ph. D.)--School of Dentistry and Dept. of Chemistry. University of Missouri--Kansas City, 2004. / "A dissertation in oral biology and chemistry." Advisor: J. David Eick. Typescript. Vita. Description based on contents viewed Feb. 23, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 137-145). Online version of the print edition.

Abstract Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from different species. In addition, LH2 has two alternatively spliced forms, LH2a and LH2b. The hydroxylysines have an important role in the formation of the intermolecular collagen crosslinks that stabilize the collagen fibrils. Some of the hydroxylysine residues are further glycosylated. In this thesis the substrate amino acid sequence specificities of the LH isoforms were analyzed using synthetic peptide substrates. The data did not indicate strict amino acid sequence specificity for the LH isoforms. However, there seemed to be a preference for some sequences to be bound and hydroxylated by a certain isoform. Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation of glucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme, possessing LH and GGT activities. The DXD-like motif, characteristic of many glycosyltransferase families, and the conserved cysteine and leucine residues in the N-terminal part of the LH3 molecule were critical for the GGT activity, but not for the LH activity of the molecule. The GGT/LH3 protein level was found to be decreased in skin fibroblasts and in the culture media of cells collected from members of a Finnish epidermolysis bullosa simplex (EBS) family, which was earlier reported to have a deficiency of GGT activity. In this study, we showed that the reduction of enzyme activity is not due to a mutation or lower expression of the LH3 gene. Our data indicate that the decreased GGT/LH3 activity in cells has an effect on the deposition and organization of the key extracellular matrix components, collagen types VI and I and fibronectin, and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as an important extracellular regulator.

Abstract Collagen molecules consist of three polypeptide chains that are coiled around each other to form a triple-helical structure. The formation of stable collagen triple helices requires the hydroxylation of proline residues catalyzed by collagen prolyl 4-hydroxylases (C-P4H). Vertebrate C-P4H is an ER-resident enzyme that consists of two catalytically active α subunits and two β subunits. Production of recombinant human collagen and gelatin could have numerous medical and industrial applications, but most recombinant systems lack the C-P4H activity. The yeast Pichia pastoris has been successfully engineered to produce stable human collagens and gelatins by co-expression of the collagen polypeptide chains with the two C-P4H subunits. This study examined the effect of deletion of the C-propeptide, or its replacement by a trimerizing foldon domain, on the assembly of type I and III collagen triple helices in P. pastoris. It was observed that the absence of the C-propeptide leads to inefficient collagen chain assembly whereas the replacement of C-propeptide with a foldon domain increased the assembly up to 3-fold. Moreover, the co-expression of α1(I) and α2(I) chains fused with foldon yielded heterotrimeric type I collagen molecules with a typical chain ratio of 2:1. As the foldon domain contains no information for collagen chain recognition, the present data indicate that the chain assembly is defined not only by the C-propeptides but also by other determinants present in the α chains. Another aspect studied here was the expression and secretion of gelatin fragments of varying size and conformation in P. pastoris. It was discovered that gelatin fragment size affects its secretion as the 90 kDa fragment was less efficiently secreted than the 45 kDa fragment. Secretion was also dependent on the fragment conformation as induction of the triple helix formation by either C-propeptide or foldon led to the accumulation of the fragments inside the yeast cells despite the presence of an efficient secretory signal. C-P4H was long assumed to exist as one type only but the cloning of several C-P4H α subunits raised questions concerning the specific roles of the C-P4H isoenzymes. The generation of mice lacking the type I C-P4H, which is regarded as the major C-P4H isoenzyme, indicated that this isoenzyme is essential for the embryonic development of the mouse. The embryos lacking type I C-P4H died at an early stage of their development due to the disruption of basement membranes. It was found that the basement membranes of the homozygous null embryos lacked type IV collagen whereas the fibrillar collagens were synthesized, although with altered morphology. The data reported here also demonstrate that the other C-P4H isoenzymes cannot compensate for the lack of type I isoenzyme.

Abstract Lysyl hydroxylases (E.C. 1.14.11.4, LHs) have three isoenzymes that are found in humans and mice, and they hydroxylate lysine residues in collagens and other proteins containing collagenous sequences. The hydroxylysines formed are crucial for the intermolecular collagen crosslinks that stabilise collagen fibres, thereby providing the stiffness and stability required by various tissues. In addition, hydroxylysines serve as attachment sites for carbohydrates, whose functions on collagen molecules are not completely understood yet. In humans, lack of LH1 causes Ehlers-Danlos syndrome (EDS) VIA, which is characterised, for example, by severe progressive kyphoscoliosis and muscular hypotonia with joint laxity. Mutations in the LH2 gene are associated with Bruck syndrome, which is characterised by fragile bones with congenital joint contractures. In the present work recombinant human lysyl hydroxylases were produced in insect cells and purified to homogeneity. Limited proteolysis revealed that LHs consist of at least three structural domains. The N-terminal domain plays no role in the lysyl hydroxylase activity, but instead, is responsible for the recently reported glucosyltransferase activity of LH3, and the galactosyltransferase activity reported here for the first time. The LH polypeptide lacking the N-terminal domain is a fully active LH with Km values identical to those of full-length enzyme. In addition, direct evidence is shown that LH2, but not LH1 or LH3, hydroxylates the telopeptide lysine residues of fibrillar collagens. All three recombinant LHs were able to hydroxylate the synthetic peptides representing the helical hydroxylation sites in types I and IV collagens, with some differences in the Vmax and Km values. In addition, all three LHs hydroxylated the collagenous domain of coexpressed type I procollagen chain to similar extend. In this study mouse lines lacking LH3 or LH1 were created and analysed. Unexpectedly, the LH3 null mice died during the embryonal period due to fragmentation of basement membranes. Type IV collagen, one of the major components in basement membranes, aggregates on its way to extracellular space and is absent from the basement membranes making them fragile. This is most probably caused by abnormal processing of type IV collagen due to decreased glucosyltransferase activity of the LH3 null embryos. The first mouse model for human EDS VIA is presented here. The LH1 null mice did not have kyphoscoliosis characteristic of EDS VIA, but showed gait abnormalities due to muscular hypotonia and possible joint laxity, as also seen in EDS VIA patients. In addition, the null mice died occasionally from aortic ruptures. Ultra structural analysis revealed degradation of smooth muscle cells and abnormal collagen fibres even in non-ruptured aortas of LH1 null mice. The hydroxylation of lysine residues and crosslinking in LH1 null mice were also abnormal, as in human EDS VIA patients. The LH1 null mouse line provides an excellent tool for analysing several aspects of human EDS VIA, including muscular hypotonia, abnormalities in collagen fibres and their crosslinking.

Abstract The Achilles tendon is the largest tendon in the human body and is affected by many diseases and is vulnerable to many forms of damage due to the heavy loads it must bear. Rupture of the Achilles tendon has become more common in recent times, with an almost four-fold increase in prevalence from 1979–1990 to 1991–2000 and a peak incidence of 19 ruptures per 100 000 of population in 1999 in our epidemiological assessment. The incidences of major complications, re-rupture and deep infection, increased along with primary ruptures, peaking in 1999. The results after successful primary repair are good in over 90% of cases, as we have shown in a randomized study and in a review of the literature, and the result after re-rupture is still good in about 70% of cases, but achieving good performance after deep infection is a highly random matter. Our retrospective survey did not identify any good results, but the deep infection cases in our randomized study showed good performance due to prompt action taken for their treatment. The best method for treating a ruptured Achilles tendon has been under debate for almost 100 years, with surgery and conservative methods advocated to equal extents. We have advocated surgical treatment as the primary choice and conservative treatment is given for selected high risk patients, for example patients with diabetes, skin problems, systemic use of corticosteroids or severe other illness. The type of surgery technique is not a straightforward choice, either, and various forms of open surgery and percutaneous techniques exist. We compared an end-to-end simple suture with the same suture augmented with one central gastrocnemius turn-over flap in a randomized series of 60 patients and found no differences with respect to subjective complaints, calf muscle strength or tendon elongation with time. The end-to-end technique is simpler and is therefore justified as the primary method of choice for the surgical repair of fresh complete Achilles tendon ruptures. The tissue composition has been shown to alter not only with time but also after repeated tearing of the tendon collagen fibres. A normal tendon is mainly composed of type I collagen, but the rupture areas express more type III collagen, which is thinner and withstands loads less effectively. Type III collagen accumulates slowly in the tendon, since its production does not increase very much, a situation that is indicative of microtrauma. Crosslinking of the fibres is important for collagen matrix properties, and we found that there is a change in the quality of crosslinking with age and that this may have role in the observed changes in tendon stiffness, as also noted in other studies. We also studied the appearance of tenascin-C at the rupture site in the Achilles tendon and at two other sites in the same tendon, but found no difference in its expression. It has been proposed that tenascin-C may take part in the tendon’s reaction to loading, but its exact function remains unknown.

Abstract Collagen XIII belongs to the subgroup of collagenous transmembrane proteins. It has a wide tissue distribution and has been localized to many sites of cell-matrix and cell-cell interaction in tissues. Biochemical and in silico analyses of collagen XIII and other collagenous transmembrane proteins revealed that the biosynthesis of this structurally varied group is characterized by a coiled-coil motif following the transmembrane domain, and these trimerization domains appear to be associated with each of the collagenous domains. The collagen XIII trimer was shown to have an interchain disulfide bond at the junction of the NC1 and COL1 domains, and several other collagenous transmembrane proteins have a pair of cysteines in the same location. Furthermore, furin cleavage at the NC1 domain can be expected in most of the proteins. Mice heterozygous for the Col13a1del transgene, encoding a mutant collagen XIII, developed clonal mature B-cell lineage lymphomas originating in the mesenteric lymph node (MLN). The incidence of disease in conventionally reared mice was 2-fold higher than for mice raised in a specific pathogen-free facility. The lymphomas often associated with large populations of macrophages and T cells. Lymphomas expressed little if any collagen XIII, suggesting that the effect of the mutation was B-cell extrinsic and likely to be associated with collagen XIII-positive tissues drained by the MLN. Studies of the small intestines of transgenic mice showed highly abnormal subepithelial basement membranes (BM), associated with heightened expression of genes involved in immune responses. These findings suggest that collagen XIII-dependent maintenance of the intestinal BM is a critical determinant of cancer susceptibility. Collagen XIII exhibited a wide tissue distribution at the protein level, and the most intense expression was found in lung. Tissues contained 1-4 collagen XIII polypeptides, their size ranging between 78 and 102 kDa. Collagen XIII staining was detected in a restricted set of blood vessels in the liver, pancreas, adrenal gland, epididymis and brain. Moreover, Col13a1del transgene expression in the absence of endogenous collagen XIII proved to be deleterious for mouse embryonal development, leading to early fetal mortality.

A fundamental study of the mechanics at the molecular scale and bridging it to the continuum level through multiscale modeling is the focus of this work. This work investigates how the material properties of nanoscale systems are influenced by the nonbonded interactions and molecular conformations. The molecular model is then bridged with the finite element model to link mechanics at nanoscale with the continuum scale. This work provides an unprecedented insight into how the interactions at the molecular scale influence mechanical properties at higher scales. Two materials are considered for the molecular modeling study: bone and Na-montmorillonite swelling clay. Bone is composed of composed of collagen molecules and hydroxyapatite in the molecular scale, which are organized into collagen fibril. The molecular dynamics study is carried out to study the nature of collagen-hydroxyapatite interface and the mechanics of collagen in bone. Furthermore, the molecular model of full-length collagen is built for the first time to show the differences in its conformation and deformation mechanism during pulling as compared to the short molecules, upon which the current understanding of is based. The mechanics of collagen is explained with the help of three-tier helical hierarchy not seen in short molecules. Two mechanisms of deformation and conformational stability of collagen are proposed: (i) interlocking gear analogy, and (ii) interplay between level-1 and level-2 hierarchies, the hydrogen bonds acting as an intermediary. The multiscale model of collagen fibril is developed by bridging nanomechanical molecular properties of collagen into the finite element model. This model shows that the molecular interactions between collagen and mineral significantly affect the mechanical response of collagen fibril. The deformation mechanism of collagen fibril and the effect of collagen crosslinks are also elucidated in this study. In recent years Na-montmorillonite has been proposed for bone regenerative medicine, besides other existing engineering applications. The molecular dynamics study of Na-montmorillonite at different levels of hydration is carried out to understand the role played by molecular interactions in the swelling behavior of Na-montmorillonite. This study greatly adds to our understanding of clay swelling, and provides important insights for modeling exfoliation and particle breakdown in clay. / NDSU Presidential Doctoral Graduate Fellowship / ND EPSCoR Doctoral Dissertation Assistantship

There are 5 distinct aspects to this study. (i) Two histological stains for collagen were compared against each other for the first time, namely Herovici's technique and picrosirius-polarization. (ii) Skin samples from embalmed cadaveric tissue from human cadavers were compared against samples taken from surgical patients. (iii) Skin samples were studied from different regions of the body to assess if dermal structure correlates with scarring potential. (iv) Skin samples were sectioned in a plane parallel to the epidermis to gain further insight into dermal structure. (v) A novel basement membrane stain was produced. Type I and type III collagen are important structural constituents of dermis and play a crucial role in wound healing. Only two traditional histological methods are thought to differentiate between them, so avoiding the need for antibodies. These were compared against each other for the first time in order to establish differences in image quality and discrimination between Type I and type III collagen. Neither technique requires antibodies, however picrosirius requires polarisation microscopy. to result in a clearer, consistently reproducible collagen staining pattern than the picrosirius method and more importantly did not require elaborate apparatus to analyze. Additionally other cellular elements were visible. Skin samples for research are often obtained from surgical excision. This clearly limits which tissues are available for comparative study to those areas operated on. Studying samples from embalmed medical school cadavers has the great advantage of studying areas of the body not routinely available from common surgical procedures. It was therefore desirable to assess whether embalmed cadaveric tissues exhibited different properties by virtue of their age and the embalming process compared to fresh surgical specimens, in order to give confidence that studies utilising the former would be equally valid. To test this, 58 skin samples from embalmed medical school cadavers were compared to skin samples from 38 fresh operative specimens. The levels of tissue preservation and processing artefacts were similar in both groups. Embalmed medical school cadavers clearly offer an opportunity to study tissue areas not routinely available during surgery. This is the first time such a comparison has been made. Many things will affect the final appearance of the scar, but the single most important determinant is the body region affected. The most common areas for unfavourable scarring, specifically keloid or hypertrophic scarring have been shown to be the ear, deltoid and sternal areas. To test the hypothesis that there is no difference in histological structure of skin that correlates to body region, comparative histology was undertaken exploring the regional variations of skin characteristics in 58 cadaveric samples. Closely comparable samples were taken from the deltoid (9), abdomen (13), sternum (10), post-auricular (5), earlobe (12) and eyelid (9). Epidermal thickness, epidermal appendage density and collagen fibre orientation were examined and qualitative structural differences were assessed for each region Skin samples were then grouped by both topographical location of the body and scarring potential. Skin samples exhibited qualitative and quantifiable regional variations in the characteristics studied. Epidermal thickness and appendage counts did not correlate with scarring potential. Both however were statistically significantly higher in skin sampled from the head compared to the trunk. Bundles of collagen fibres in the reticular dermis were grouped according to their orientation in relation to the coronal plane; either parallel, oblique or perpendicular. The ratio of oblique to parallel fibres was statistically significantly higher in body areas with poorer scarring prognosis. This corresponds to a more disorganised arrangement of collagen fibres in these areas. Further qualitative understanding of dermal collagen fibres came from perpendicular to conventional histological samples. This new method stained basement membranes purple, cytoplasm was stained greenish-brown and nuclei dark brown. Collagen fibres were either thin and blue or thick and green. This method was compared to PAS staining and although required more preparative steps allows greater identification of other cellular structures.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 1977 / Includes bibliographical references. / by Frederick H. Silver. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Mechanical Engineering