Connexin 43 (Cx43) is a gap junction protein that enables direct cell-to-cell communication in many tissues of the body and stressful environments such as injury and hypoxia affect its expression. In addition to affecting Cx43 expression, hypoxia also retards wound-healing rates. Using rat corneal organ cultures, this thesis aims to define hypoxia's role in the expression and localization of Cx43 in wounded corneas. Tissue samples were stained immunohistochemically at three different time points after injury, 2.5 hours, 5 hours, and 18 hours. It was determined that under hypoxic conditions, Cx43 is more highly expressed than under normoxic conditions after injury. While Cx43 expression follows the same spatial pattern throughout the healing process in the cornea under both normoxic and hypoxic conditions, the pattern is delayed under hypoxic conditions. These results provide a possible reason why wound closure is delayed in low oxygen environments.

No description available.

Substances of natural and synthetic origin were studied using analytical, bioassay guided isolation, metabolomics and medicinal chemistry techniques. In a section focused on the plant family Marcgraviaceae, a validated method for the quantification of six pentacyclic triterpenes (α and β Amyrin lupeol, ursolic acid, betulin and betulinic acid) in the Souroubea spp was developed. Quantification of the triterpenes in the crude extracts was achieved using HPLC-APCI mass selective detection. The calibration curves for the five triterpenes evaluated were highly linear (r2 >0.993) and percentage recovery from spiked samples were greater than 94% for all compounds. The LOD for betulinic acid was 0.01 µg for betulinic acid on column and LOQ was 0.03 µg. The method was successfully applied to 41 crude extracts from leaf and stem of Souroubea spp, from two locations in Costa Rica. The method is suitable for quality control of raw materials used in the manufacture of natural health products. The use of modern metabolomic techniques, UHPLC-QTOF allowed the identification of five putative makers that can potentially be used in distinguishing between the two Souroubea species. The validated method was used in the quantification of the above triterpenes in a total of thirteen Marcgraviaceae species collected in Costa Rica. It was established that betulinic acid and β- Amyrin could be used as makers for this family of tropical vines. These same thirteen plants extracts were evaluated in antifungal and quorum sensing inhibition bioassays. Marcgravia nervosa was the only species that showed significant activity in both bioassays. Bioassay guided fractionation of the crude ethanolic extract of M. nervosa led to the identification of 2-methoxynaphthoquinone as the bioactive compound responsible for the bioactivity. The crude leaf ethanolic extract from M. nervosa showed a significant inhibition of QS comparable or somewhat better than D. pulchra extracts with the M. nervosa extract showing stronger inhibiting QS with a halo of 21.8mm, more than D. pulcra extracts which generated a halo of 15.9mm. The active quinone has a MIC of 85 µM against Saccharomyces cerevisiaBY4741 (haploid) and 100 µM against Saccharomyces cerevisiae BY4743 (diploid) compared to berberine (positive control) with a MIC 600 µM for both strains. This quinone is not present in any of the other twelve species of Marcgraviaceae available to us. In work focusing on organic synthesis, a total of 57 semi-synthetic derivatives of dillapiol, safrol and piperonal were prepared and evaluated for their inhibitory activity in a CYP 3A4 bioassay to assess their potential use as pesticide synergists. The synergistic activity of dillapiol has been improved 45 fold; analog 31 has an IC50 = 0.2 µM compared with dillapiol IC50= 9.18 µM. A number of other compounds structurally related to 31 showed similar levels of activity. A screening of a compound library identified the amino sulfoxide 3 as a potential lead for the design of a selective connexin blocker with potential application in the treatment of spinal cord injuries. The use of X-ray crystallography permitted the correction of the original structure assigned to 3. Once the structure was corrected a total of 6 analogs were prepared. Compound 3 has the highest inhibition of GJIC whereas compound 8 and compound 2, reduced anionic hemi-channel activity. Compound 2 also reduced the cationic activity of the hemi-channels.

国立情報学研究所で電子化したコンテンツを使用している。 / 国立情報学研究所で電子化したコンテンツを使用している。

Zugl.: Tübingen, Universiẗat, Diss., 2008.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2000. / Includes bibliographical references (leaves 174-192). Also available online through Digital Dissertations.

Substances of natural and synthetic origin were studied using analytical, bioassay guided isolation, metabolomics and medicinal chemistry techniques. In a section focused on the plant family Marcgraviaceae, a validated method for the quantification of six pentacyclic triterpenes (α and β Amyrin lupeol, ursolic acid, betulin and betulinic acid) in the Souroubea spp was developed. Quantification of the triterpenes in the crude extracts was achieved using HPLC-APCI mass selective detection. The calibration curves for the five triterpenes evaluated were highly linear (r2 >0.993) and percentage recovery from spiked samples were greater than 94% for all compounds. The LOD for betulinic acid was 0.01 µg for betulinic acid on column and LOQ was 0.03 µg. The method was successfully applied to 41 crude extracts from leaf and stem of Souroubea spp, from two locations in Costa Rica. The method is suitable for quality control of raw materials used in the manufacture of natural health products. The use of modern metabolomic techniques, UHPLC-QTOF allowed the identification of five putative makers that can potentially be used in distinguishing between the two Souroubea species. The validated method was used in the quantification of the above triterpenes in a total of thirteen Marcgraviaceae species collected in Costa Rica. It was established that betulinic acid and β- Amyrin could be used as makers for this family of tropical vines. These same thirteen plants extracts were evaluated in antifungal and quorum sensing inhibition bioassays. Marcgravia nervosa was the only species that showed significant activity in both bioassays. Bioassay guided fractionation of the crude ethanolic extract of M. nervosa led to the identification of 2-methoxynaphthoquinone as the bioactive compound responsible for the bioactivity. The crude leaf ethanolic extract from M. nervosa showed a significant inhibition of QS comparable or somewhat better than D. pulchra extracts with the M. nervosa extract showing stronger inhibiting QS with a halo of 21.8mm, more than D. pulcra extracts which generated a halo of 15.9mm. The active quinone has a MIC of 85 µM against Saccharomyces cerevisiaBY4741 (haploid) and 100 µM against Saccharomyces cerevisiae BY4743 (diploid) compared to berberine (positive control) with a MIC 600 µM for both strains. This quinone is not present in any of the other twelve species of Marcgraviaceae available to us. In work focusing on organic synthesis, a total of 57 semi-synthetic derivatives of dillapiol, safrol and piperonal were prepared and evaluated for their inhibitory activity in a CYP 3A4 bioassay to assess their potential use as pesticide synergists. The synergistic activity of dillapiol has been improved 45 fold; analog 31 has an IC50 = 0.2 µM compared with dillapiol IC50= 9.18 µM. A number of other compounds structurally related to 31 showed similar levels of activity. A screening of a compound library identified the amino sulfoxide 3 as a potential lead for the design of a selective connexin blocker with potential application in the treatment of spinal cord injuries. The use of X-ray crystallography permitted the correction of the original structure assigned to 3. Once the structure was corrected a total of 6 analogs were prepared. Compound 3 has the highest inhibition of GJIC whereas compound 8 and compound 2, reduced anionic hemi-channel activity. Compound 2 also reduced the cationic activity of the hemi-channels.

Indiana University-Purdue University Indianapolis (IUPUI) / Old age is associated with increased bone fragility and risk of fracture as a result of skeletal alterations, including low bone density and cortical thinning. Further, apoptotic osteocytes accumulate in old mice and humans. We have previously shown that mice lacking osteocytic connexin (Cx) 43 (Cx43ΔOt) exhibit a phenotype similar to that of the aging skeleton, with elevated osteocyte apoptosis and an associated increase in osteoclastogenesis. These findings suggest that osteocyte apoptosis results in the release of factors that recruit osteoclasts to bone surfaces close to areas that contain apoptotic osteocytes. However, the specific chemotactic signals, the events mediating their release, and the mechanisms of their action remain unknown. Consistent with this notion, we also found that HMGB1 released by Cx43-deficient (Cx43def) MLO-Y4 osteocytic cells enhances osteoclastogenesis in part by increasing osteocytic RANKL, which promotes osteoclastogenesis, and, at the same time, directly stimulating osteoclastogenesis. Further, expression of the pro-survival microRNA (miR), miR21, is low in Cx43def cells and bones from old female mice, and low miR21 levels increase osteocyte apoptosis. However, surprisingly, mice lacking miR21 (miR21ΔOt) have decreased osteoclast number and activity even under conditions of elevated osteocyte apoptosis; suggesting that osteocytic miR21 may mediate osteoclast precursor recruitment/survival induced by apoptotic osteocytes. However, whether HMGB1/miR21 are released by osteocytes, and if the HMGB1 receptors, receptor for advanced glycation end products (RAGE) and/or tolllike receptor (TLR4) are involved in osteoclast recruitment in Cx43ΔOt and old mice is unknown. The overall objectives of this series of studies were to elucidate the mechanisms

No description available.