The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on October 7, 2009) Thesis advisor: Dr. Kathy L. Sharpe-Timms. Includes bibliographical references.

Thesis (Ph. D.)--Washington State University, December 2009. / Title from PDF title page (viewed on Sept. 9, 2009). "Program in Neuroscience." Includes bibliographical references.

Thesis (M.Sc.)--York University, 2004. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 121-137). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ99345

Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Anatomy and Neurobiology . Bibliography: leaves 108 - 125. Also available online.

Thesis (Ph. D.)--Washington State University, December 2006. / Includes bibliographical references (p. 100-110).

This study had two aspects. The first was the production and purification of TIMP-l. The second was a series of biophysical studies of TIMP-l and a TIMP-l derived peptide. A monoclonal antibody affinity column was developed and used to purifY large quantities of human TIMP-l for further experiments. Two E.coli expression systems were studied to determine whether they would be suitable for large scale production of recombinant protein. In the first system TIMP-I was to be secreted as a fusion protein which could be cleaved, leaving a free N-terminus. It was discovered that it was not possible to cleave off the fusion protein. In the second system, the protein was secreted, without additions to the periplasm. Although active protein, with the correct N-tenninus, was obtained, the yields were too low to be of use for large scale expression. Secondary structure analysis by CD and FTIR showed TIMP-l to be a mostly f3- sheet protein (approaching 50%) with around 20% a-helix. A temperature study using these techniques found that little change occurs until temperatures of over 60°C where the protein aggregates. The small changes appear to be a general loosening of the structure. In analyses of the surface of TIMP-l, additional carbohydrate was identified (other than the two N-linked chains) using Con-A probing of Western blots. TIMP-l purified from WI-38 foetal lung fibroblast cells can be separated into two pools by Concanavalin A-Sepharose chromatography. These two pools were found to have a different set of pIs and a different monosaccharide composition. The use of NMR paramagnetic probes identified a hydrophobic region exposed on the surface of TIMP-I. This region probably includes a tyrosine residue and either a tryptophan or phenylalanine. The presence of an exposed hydrophobic region was also shown in binding studies using the fluorescent probe ANS. These studies identified a single, low affinity binding site. An additional study with the N-terminal fragment of type-I collagenase found no binding sites on the enzyme, but a change in fluorescence occurred when TIMP-I was present. A peptide was designed based on the N-terminal sequence of TIMP-I. High homology, susceptibility to mutation and an interesting resemblance to the Bowman-Birk family of inhibitors suggested that this peptide might be inhibitory. It was found to have only a weak inhibitory activity against gelatinase. NMR studies of this peptide in water showed a large number of conformers as a result of stabilisation of the cis isomer of its proline residues. This preference for the cis form was retained for one proline in the solvent, TFE. Preliminary NMR studies were also carried out which concluded that TIMP-I should be suitable for further structural studies using isotopic labelling.

Orientador: Ana Paula de Souza Pardo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-15T12:46:18Z (GMT). No. of bitstreams: 1 Planello_AlineCristiane_M.pdf: 940083 bytes, checksum: 36ed3b0b3b0d10c144805116e08917dd (MD5) Previous issue date: 2010 / Resumo: Objetivo: As Metaloproteinases da Matriz ( MMPs) são enzimas que degradam a matriz extracelular (MEC) e tem sido associadas às desordens temporomandibulares (DTM). Nós investigamos a freqüência dos -1607 1G/2G MMP1 polimorfismo (rs1799750), -1171 6A/5A MMP3 polimorfismo (rs3025058) e -1562 C/T MMP9 polimorfismo (rs3918242) em indivíduos com sinais de degeneração da ATM, diagnosticados por exame de imagem, a fim de analisar a associação desses polimorfismos e a DTM. Métodos: A população estudada foi composta por 115 indivíduos diagnosticados por exame de imagem (grupo DTM) e 117 controles. Os polimorfismos genéticos foram determinados por PCR/RFLP. Resultados: A freqüência do genótipo 2G/2G no gene MMP1 foi significantemente mais alta no grupo DTM do que no grupo Controle (p = 0.008). O genótipo 2G/2G no grupo DTM mostrou um risco aumentado para a DTM com um OR = 2.25 ( 95% IC = 1.26 - 3.99) quando comparado com os genótipo 1G/2G e 1G/1G. A freqüência dos alelos do gene MMP1 não mostrou diferença significativa entre os grupos (p > 0.05). A distribuição dos genótipos e alelos dos genes MMP3 e MMP9 não mostrou diferença significativa (p > 0.05). Conclusão: Nossos resultados mostram a associação entre o polimorfismo -1607 MMP1 e a suscetibilidade à DTM / Abstract: Objective. Matrix metalloproteinases (MMPs) degrade extracellular matrix components and have been implicated to play an important role in temporomandibular joint disorder (TMD). We investigated the frequency of -1607 1G/2G MMP1 polymorphism (rs1799750), -1171 6A/5A MMP3 polymorphism (rs3025058) and -1562 C/T MMP9 polymorphism (rs3918242) in individuals with TMJ degeneration diagnosed by image exam in order to analyze the association of these MMPs polymorphisms and TMD. Methods. The studied population comprised 115 TMD individuals diagnosed by image exam and 117 healthy controls. Genotypes were determined using polymerase chain reaction/Restriction fragment length polymorphism PCR/RFLP. Results. The MMP1 2G/2G genotype was significantly higher in the TMD group than in the Control group (p = 0.008). The genotype 2G/2G in the TMD group showed an increased risk to TMD with an OR = 2.25 (95% CI = 1.26 - 3.99) when compared with 1G/2G and 1G/1G genotypes. Analysis of MMP1 allele frequencies showed no significant difference (p > 0.05). The MMP3 and MMP9 genotypes distribution and alleles frequency did not differ between the groups (p > 0.05). Conclusion. Our results report the association of -1607 MMP1 gene polymorphism and increased risk to TMD / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental

Expression of membrane-type matrix metalloproteinases (MT) and tissue inhibitors of matrix metalloproteinases (TIMP) was evaluated in sheep embryos and uterus during the pre- and peri-implantation periods. Embryos and uterine samples were surgically collected from ewes on days 9, 11, 13, and 15 of pregnancy (n=3 ewes/day) and of the estrous cycle (n=2 ewes/day). Total RNA was extracted and RT-PCR were performed using primers specifically designed from published human, mouse, and bovine complete cDNA sequences for MT-1, -2, -3, and -5, and TIMP-1, -2 and -3. Multiplex PCR were performed on uterine samples for each gene at optimal cycles and temperatures with 18S rRNA as the internal standard. For embryos, PCR were conducted for 40 cycles at optimal temperatures. MT-1, -2, -3, and -5 were observed in pregnant and nonpregnant uterus during all days of collection. No difference (P>0.10) was observed in MT-1 or -2 expression due to day of collection. However, pregnant uterus expressed more (P=0.096) MT-1 than nonpregnant uterus, whereas expression of MT-2 was greater (P<0.05) in nonpregnant compared to pregnant uterus. No differences (P>0.10) in MT-3 expression were observed due to pregnancy status, however Day 9 and 11 expressed more MT-3 than Day 15. Uterine MT-5 expression was not different (P>0.10) between pregnant and nonpregnant females, however Day 15 uterus expressed less (P<0.05) MT-5 then Day 11 and 13 uteri. TIMP-1 expression was greater (P<0.05) in pregnant compared to nonpregnant uterus, but did not differ (P>0.10) by day of collection. TIMP-2 did not differ (P>0.10) by pregnancy status or day of collection but the interaction was significant (P<0.05). TIMP-2 expression was greatest in Day 9 pregnant uterus and least in Day 9 nonpregnant uterus. No difference (P>0.10) was observed in expression of TIMP-3 due to day of collection or pregnancy status. Embryos expressed MT-3 and -5 during Days 9-15 of development, however, MT-1 and -2 were not detected. The presence of MT and TIMP in the endometrium suggests these proteins may play important roles in regulating extracellular matrix degradation and activating other matrix metalloproteinases for endometrial remodeling and preparation for implantation. Embryonic MT may participate in the processes of embryonic expansion, elongation and attachment. / Graduation date: 2002

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Matrix metalloproteinases (MMPs) are known for their role in extracellular matrix remodeling, but their role in regulating intracellular immune cell function is unknown. We reported that MMP inhibition down regulated T cell proliferation in response to alloantigens and autoantigens; but the direct role of MMP involvement in T cell activation has not been reported. Methods: MMP deficient or MMP sufficient wild-type CD4+ or CD8+ T cells from C57BL/6 mice were treated with SB-3CT, a specific inhibitor of MMP2 and MMP9, stimulated with anti-CD3 Ab, alone, or with IL-2 or CD28. Cellular activation and cytokine profiles were examined. A mouse model of antigen specific T cell mediated lung injury was used to examine MMP inhibition in antigen-specific T cell mediated lung injury. Results: SB-3CT (1-25μM) induced dose-dependent reductions in anti-CD3 Ab-induced proliferation (p<0.0001). Compared to wild-type, MMP9-/- CD4+ and CD8+ T cells proliferated 80-85% less (p<0.001) in response to anti-CD3 Ab. Compared to untreated or wild-type cells, anti-CD3 Ab-induced calcium flux was enhanced in SB-3CT-treated or MMP9-/- CD4+ and CD8+ T cells. Cytokine transcripts for IL-2, TNF-α and IFN-γ were reduced in both CD4+ and CD8+ MMP9-/- T cells, as well as in SB3CT treated CD4+ T cells. MMP inhibition dampened antigen-specific T cell mediated lung injury. Conclusions: Although known to be functional extracellularly, the current data suggest that MMPs function inside the cell to regulate intracellular signaling events involved in T cell activation. T cell targeted MMP inhibition may provide a novel approach of immune regulation in the treatment of T cell-mediated diseases. - David S. Wilkes, M.D., Chair.

No description available.