Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [118]-127).

Thesis (Ph. D.)--University of Hong Kong, 1998. / Cover title. Includes bibliographical references (leaves 180-209).

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Elliot L. Chaikof; Committee Member: Ajit Yoganathan; Committee Member: Larry McIntire; Committee Member: Marc Levenston; Committee Member: Mark Allen. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2005. / Vita. Includes bibliographical references.

Made available in DSpace on 2014-08-13T14:50:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-09-12Bitstream added on 2014-08-13T18:01:10Z : No. of bitstreams: 1 000736306_20140912.pdf: 355338 bytes, checksum: 8b660dc8c366e3de685e2a2e9ba80c6b (MD5) Bitstreams deleted on 2014-10-03T16:24:43Z: 000736306_20140912.pdf,Bitstream added on 2014-10-03T16:27:40Z : No. of bitstreams: 2 000736306_20140912.pdf.txt: 34366 bytes, checksum: a356e133fe540b7d9ac63b7804a6a7f2 (MD5) 000736306.pdf: 3412132 bytes, checksum: 10ad56d9db7be90218af0d380682ebd0 (MD5) Bitstreams deleted on 2014-10-03T16:33:17Z: 000736306.pdf,Bitstream added on 2014-10-03T16:43:31Z : No. of bitstreams: 2 000736306_20140912.pdf.txt: 34366 bytes, checksum: a356e133fe540b7d9ac63b7804a6a7f2 (MD5) 000736306.pdf: 3412132 bytes, checksum: 10ad56d9db7be90218af0d380682ebd0 (MD5) Bitstreams deleted on 2014-10-03T16:48:57Z: 000736306.pdf,Bitstream added on 2014-10-03T16:49:48Z : No. of bitstreams: 1 000736306.pdf: 3412132 bytes, checksum: 10ad56d9db7be90218af0d380682ebd0 (MD5) Bitstreams deleted on 2014-10-27T11:47:12Z: 000736306.pdf,Bitstream added on 2014-10-27T11:48:06Z : No. of bitstreams: 1 000736306.pdf: 3412132 bytes, checksum: 10ad56d9db7be90218af0d380682ebd0 (MD5) Bitstreams deleted on 2014-11-14T12:16:56Z: 000736306.pdf,Bitstream added on 2014-11-14T12:17:44Z : No. of bitstreams: 1 000736306.pdf: 3412190 bytes, checksum: 064d4a5b064f6771c20a0ed9daeb980f (MD5) / O objetivo geral deste trabalho foi avaliar a citotoxicidade transdentinária da carbodiimida (EDC), bem como sua influência na degradação do colágeno dentinário e na estabilidade da união resina-dentina. No estudo 1, células MDPC- 23 foram plantadas na superfície pulpar de discos de dentina e a superfície oclusal foi tratada por 60s com uma das seguintes soluções: sem tratamento; EDC 0,1M; 0,3M ou 0,5M; glutaraldeído 5% (GA); tampão Sorensen ou H2O2 29%. A viabilidade e a morfologia celular foram analisadas pelos testes de MTT, Live/dead, produção de proteína total (PT), de colágeno e MEV. Os dados foram analisados pelos testes de Kruskal-Wallis e Mann-Whitney (p<0,05). O GA promoveu aumento do metabolismo celular. A morte por necrose e a morfologia celular não foram influenciadas pelos agentes cross-linkers. Não houve redução na produção de PT e colágeno após 7 dias. Para o estudo 2, espécimes de dentina foram completamente desmineralizados e a variação do módulo de elasticidade (E), inibição de MMP, perda de massa, liberação de hidroxiprolina (HYP) e degradação térmica do colágeno (DTC) foram analisados após tratamento com uma das seguintes soluções por 30s ou 60s: água deionizada (controle); EDC 0,5M; EDC 1M; EDC 2M e GA 10%. Os dados referentes ao E, atividade de MMP e liberação de HYP foram submetidos aos testes de Wilcoxon e Kruskal- Wallis ou Mann-Whitney. Os valores de perda de massa e DT foram analisados pelos testes de ANOVA e Tukey (p<0,05). Os melhores resultados quanto ao E foram observados para o GA. Todos os cross-linkers reduziram a atividade de MMP e a liberação de HYP e aumentaram a temperatura de DT do colágeno. No estudo 3, sessenta palitos de dentina foram divididos em 6 grupos de acordo com a solução de tratamento: água deionizada (controle); EDC 0,1M; EDC 0,5M; EDC 0,5M + HEMA 35%; proantocianidina 5% (PA) ou clorexidina (CHX) 2%. Após... / The purpose of this study was to evaluate the trandentinal cytotoxicity of carbodiimide (EDC), as well as its influence on dentinal collagen degradation and stability of resin-dentin bonds. In the first experiment, MDPC-23 cells were seeded on the pulp surface of the disks and one of the following solutions was applied on the occlusal surface for 60s: no treatment (negative control), 0.1M, 0.3M or 0.5M EDC; 5% glutaraldehyde (GA); Sorensen buffer or 29% hydrogen peroxide (positive control). Cell viability and morphology were analyzed by MTT, Live/Dead assays, total protein (TP) and collagen production and SEM. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (p<0.05). Only GA increased cellular metabolism. Cell death by necrosis and cell morphology were not affected by the cross-linker agents. There was no reduction in TP and collagen production after 7 days. For the second experiment, dentin beams were completely demineralized and the variation in elastic modulus (E), MMP activity, dry mass loss, hydroxyproline release (HYP) and collagen thermal degradation (CTD) were analyzed after the dentin treatment for 30s or 60s with the following solutions: water; 0.5M; 1M or 2M EDC and 10% GA. Data from E and MMP activity and HYP release were submitted to Wilcoxon and Kruskal-Wallis or Mann-Whitney tests. Dry mass loss and CTD data were analyzed by ANOVA and Tukey’s tests (p>0.05). GA group obtained the highest E values. All cross-linking agents decreased MMP activity and HYP release and increased CTD. In the third experiment, sixty dentin beams were randomly divided into 6 groups according to the treatment solution: deionized water (control), 0.1M EDC, 0.5M EDC, 0.5M EDC+35% HEMA, 5% proanthocyanidin (PA) or 2% chlorhexidine (CHX). The beams were acid etched and treated for 60s. The total MMP activity was analyzed by a colorimetric assay (Sensolyte®). Data were expressed as absorbance values...

Orientador: Josimeri Hebling / Banca: Fabio Duparte Nacimento / Banca: Linda Wang / Banca: Elisa Maria Aparecida Giro / Banca: Gelson Luis Adabo / Resumo: O objetivo geral deste trabalho foi avaliar a citotoxicidade transdentinária da carbodiimida (EDC), bem como sua influência na degradação do colágeno dentinário e na estabilidade da união resina-dentina. No estudo 1, células MDPC- 23 foram plantadas na superfície pulpar de discos de dentina e a superfície oclusal foi tratada por 60s com uma das seguintes soluções: sem tratamento; EDC 0,1M; 0,3M ou 0,5M; glutaraldeído 5% (GA); tampão Sorensen ou H2O2 29%. A viabilidade e a morfologia celular foram analisadas pelos testes de MTT, Live/dead, produção de proteína total (PT), de colágeno e MEV. Os dados foram analisados pelos testes de Kruskal-Wallis e Mann-Whitney (p<0,05). O GA promoveu aumento do metabolismo celular. A morte por necrose e a morfologia celular não foram influenciadas pelos agentes cross-linkers. Não houve redução na produção de PT e colágeno após 7 dias. Para o estudo 2, espécimes de dentina foram completamente desmineralizados e a variação do módulo de elasticidade (E), inibição de MMP, perda de massa, liberação de hidroxiprolina (HYP) e degradação térmica do colágeno (DTC) foram analisados após tratamento com uma das seguintes soluções por 30s ou 60s: água deionizada (controle); EDC 0,5M; EDC 1M; EDC 2M e GA 10%. Os dados referentes ao E, atividade de MMP e liberação de HYP foram submetidos aos testes de Wilcoxon e Kruskal- Wallis ou Mann-Whitney. Os valores de perda de massa e DT foram analisados pelos testes de ANOVA e Tukey (p<0,05). Os melhores resultados quanto ao E foram observados para o GA. Todos os cross-linkers reduziram a atividade de MMP e a liberação de HYP e aumentaram a temperatura de DT do colágeno. No estudo 3, sessenta palitos de dentina foram divididos em 6 grupos de acordo com a solução de tratamento: água deionizada (controle); EDC 0,1M; EDC 0,5M; EDC 0,5M + HEMA 35%; proantocianidina 5% (PA) ou clorexidina (CHX) 2%. Após... / Abstract: The purpose of this study was to evaluate the trandentinal cytotoxicity of carbodiimide (EDC), as well as its influence on dentinal collagen degradation and stability of resin-dentin bonds. In the first experiment, MDPC-23 cells were seeded on the pulp surface of the disks and one of the following solutions was applied on the occlusal surface for 60s: no treatment (negative control), 0.1M, 0.3M or 0.5M EDC; 5% glutaraldehyde (GA); Sorensen buffer or 29% hydrogen peroxide (positive control). Cell viability and morphology were analyzed by MTT, Live/Dead assays, total protein (TP) and collagen production and SEM. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (p<0.05). Only GA increased cellular metabolism. Cell death by necrosis and cell morphology were not affected by the cross-linker agents. There was no reduction in TP and collagen production after 7 days. For the second experiment, dentin beams were completely demineralized and the variation in elastic modulus (E), MMP activity, dry mass loss, hydroxyproline release (HYP) and collagen thermal degradation (CTD) were analyzed after the dentin treatment for 30s or 60s with the following solutions: water; 0.5M; 1M or 2M EDC and 10% GA. Data from E and MMP activity and HYP release were submitted to Wilcoxon and Kruskal-Wallis or Mann-Whitney tests. Dry mass loss and CTD data were analyzed by ANOVA and Tukey's tests (p>0.05). GA group obtained the highest E values. All cross-linking agents decreased MMP activity and HYP release and increased CTD. In the third experiment, sixty dentin beams were randomly divided into 6 groups according to the treatment solution: deionized water (control), 0.1M EDC, 0.5M EDC, 0.5M EDC+35% HEMA, 5% proanthocyanidin (PA) or 2% chlorhexidine (CHX). The beams were acid etched and treated for 60s. The total MMP activity was analyzed by a colorimetric assay (Sensolyte®). Data were expressed as absorbance values... / Doutor

Abstract Lysyl hydroxylase (E.C. 1.14.11.4, protocollagen-lysine 2-oxoglutarate 5-dioxygenase, PLOD) catalyses the formation of hydroxylysine in collagens and other proteins with collagen-like sequences. The hydroxylysine residues participate in the formation of collagen crosslinks and serve as attachment sites for carbohydrate units. The importance of lysine hydroxylation is demonstrated by the critical manifestations found in patients with the type VI variant of the Ehlers-Danlos syndrome, which is caused by a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate. The binding site for the C-5 carboxyl group of 2-oxoglutarate is characterized here by site-directed mutagenesis. Two conserved and one non-conserved amino acid residues at the possible binding site in human lysyl hydroxylase 1 were converted individually to alanine or lysine and the mutant polypeptides were expressed in insect cells. Mutation of arginine-700 to alanine inactivated the enzyme completely, whereas mutation of the other two residues had only a minor effect. In addition, the Km of the arginine-700 to lysine mutant polypeptide for 2-oxoglutarate was increased 10-fold. The results thus indicate that this conserved arginine is the residue that binds the C-5 carboxyl group of 2-oxoglutarate in lysyl hydroxylases. A novel human lysyl hydroxylase isoenzyme, termed lysyl hydroxylase 3, was identified, cloned and characterized here. The overall amino acid sequence identity between the novel human lysyl hydroxylase isoenzyme and the other human lysyl hydroxylase isoenzymes is about 60%. The highest expression levels of the mRNA for lysyl hydroxylase 3 among the tissues studied were found in the placenta, pancreas and heart. The novel isoenzyme was expressed as a recombinant protein in insect cells, and the protein was shown to function as a lysyl hydroxylase in vitro hydroxylation experiments using short synthetic peptides as substrates. No differences in catalytic properties were found between the recombinant lysyl hydroxylases 3 and 1. The structure of the human gene for lysyl hydroxylase 3 was determined in the last part of this work. The gene is shown to be only 11.6 kb in size and to contain 19 exons. Transcription was found to be initiated at multiple sites, and the introns contained 15 full-length Alu retroposons or partial Alu fragments of more than 100 bp. The present characterization of the exon-intron organization of the gene will provide a basis for further studies to determine whether there is any genetic disease that is attributable to mutations in this gene.

Abstract Collagens IX, a non-fibrillar collagen, and XI, a fibrillar collagen, are minor components of cartilage collagen fibrils, which form a supportive meshwork in the cartilage extracellular matrix (ECM). Collagens IX and XI are known to be present also in other tissues, including the vitreous body of the eye, the intervertebral disc, the inner ear, and various tissues during embryonic development. Collagen IX is suggested to act as a macromolecular bridge between collagen fibrils and other ECM molecules, and it may be important for the cohesive and compressive properties of cartilage, as well as the long-term stability of articular cartilage. Collagen XI is speculated to have a role in regulating the fibril diameter, and it may participate in interactions with other ECM components. However, the role of neither collagen IX nor XI has been confirmed yet. As important but minor components of the cartilage ECM, collagens IX and XI are excellent candidates for relatively mild chondrodysplasias and even milder disease phenotypes involving cartilaginous tissues, such as non-syndromic hearing loss. There are in fact many reports describing defects in the genes for collagens IX and XI in patients with a variety of chondrodysplasias, including multiple epiphyseal dysplasia, Stickler syndrome, Marshall syndrome and otospondylomegaepiphyseal dysplasia. In order to screen the minor cartilage collagen genes for mutations, it is essential to know their gene structures. Therefore, the complete structures of the human COL9A1, COL9A2 and COL11A2 genes were characterized in this study. Also, to facilitate the analysis of the 5' region of the COL11A2 gene, the cDNA and partial genomic structure of the mouse Col11a2 gene were defined. The information obtained in this study was utilized in the mutation analysis of a family with non-ocular Stickler syndrome. The COL11A2 gene was analyzed with conformation sensitive gel electrophoresis (CSGE) and sequencing, and a heterozygous single-nucleotide mutation causing a premature termination codon was found in the affected family members. Studying the effect of the mutation on the RNA revealed that the nonsense mutation caused the skipping of a 54-bp exon, presumably through a pathway called nonsense-associated altered splicing.

Abstract Collagen prolyl 4-hydroxylase is the key enzyme in the biosynthesis of collagens, a family of extracellular matrix proteins. Vertebrate collagen prolyl 4-hydroxylases are α2β2 tetramers, the β subunit being identical to the multifunctional protein disulphide isomerase (PDI). Several isoforms of the catalytic α subunit have been identified in various organisms. Prolyl 4-hydroxylases have also been isolated from plants, where they hydroxylate proline-rich structural glycoproteins of the cell walls. The structural and functional properties of the peptide-substrate-binding domain of human collagen prolyl 4-hydroxylase are characterized here. Data obtained from NMR studies indicate that the domain consists of five α helices and one short β strand, this structure being quite different from those of other proline-rich peptide-binding modules. Several residues involved in the binding of a short synthetic peptide were also identified by NMR. Kd values for the binding of several synthetic peptides to the α(I) and α(II) domains were determined by surface plasmon resonance and isothermal calorimetry, and the results indicated that the binding properties of the type I and type II collagen prolyl 4-hydroxylase tetramers can mainly be explained by the binding of peptides to this domain rather than to the catalytic domain. The peptide-substrate-binding domain of human type I collagen prolyl 4-hydroxylase was also crystallized. The crystals were well ordered and diffracted to at least 3 Å, the asymmetric unit most probably containing a domain dimer. The genome of Arabidopsis thaliana was found to encode at least six putative prolyl 4-hydroxylase polypeptides, one of which was cloned and characterized here as a recombinant protein. All the catalytically critical residues identified in animal prolyl 4-hydroxylases were also conserved in this plant prolyl 4-hydroxylase, and their mutagenesis led to inactivation of the enzyme. The recombinant plant enzyme was effective in hydroxylating poly(L-proline) and several synthetic proline-rich peptides. Surprisingly, contrary to previous reports on plant prolyl 4-hydroxylases, the collagen-like peptides were found to be good substrates, the enzyme preferentially hydroxylating prolines in the Y positions of the -X-Y-Gly- triplets, thus resembling the vertebrate collagen prolyl 4-hydroxylases even in this respect. The recombinant plant prolyl 4-hydroxylase also hydroxylated peptides representing the N and C-terminal hydroxylation sites present in the hypoxia-inducible transcription factor α. The fact that these peptides contain only one proline residue indicated that a poly(L-proline) type II conformation was not required for hydroxylation.

Please read the abstract in the section 00front of this document / Thesis (PhD(Food Science))--University of Pretoria, 2005. / Food Science / unrestricted