At present, vascular endothelial cell (VEC) apoptosis induced by deprivation of fibroblast growth factor-2 (FGF-2) and serum has been well studied. But how to trigger VEC apoptosis in the presence of FGF-2 and serum is not well known. To address this question, in this study, the effects of safrole oxide on angiogenesis and VEC growth stimulated by FGF-2 were investigated. The results showed that safrole oxide inhibited angiogenesis and induced VEC apoptosis in the presence of FGF-2 and serum. To understand the possible mechanism of safrole oxide acting, we first examined the phosphorylation of Akt and the activity of nitric oxide synthase (NOS); secondly, we analyzed the expressions and distributions of Fas and P53; then we measured the activity of phosphatidylcholine specific phospholipase C (PC-PLC) in the VECs treated with and without safrole oxide. The results showed that this small molecule obviously suppressed Akt phosphorylation and the activity of NOS, and promoted the expressions of Fas and P53 markedly. Simultaneously, Fas protein clumped on cell membrane, instead of homogenously distributed. The activity of PC-PLC was not changed obviously. The data suggested that safrole oxide effectively inhibited angiogenesis and triggered VEC apoptosis in the presence of FGF-2 and serum, and it might perform its functions by suppressing Akt/NOS signal pathway, upregulating the expressions of Fas and P53 and modifying the distributing pattern of Fas in VEC. This finding provided a powerful chemical probe for promoting VEC apoptosis during angiogenesis stimulated by FGF-2.

Indiana University-Purdue University Indianapolis (IUPUI) / Dynamic changes to the extracellular matrix (ECM) impact many cell fate pro- cesses. The ECM can experience changes in sti ness as well as changes in composi- tion in response to injury, development, and diseases. To better understand the role that these dynamic processes have on the cells residing within the environment, re- searchers have turned towards 4-dimensional (4D) hydrogel designs. These 4D hydro- gels re-capitulate not only 3-dimensional (3D) matrix architectures, but also temporal changes in the physicochemical properties. The goal of this thesis was to design a unify chemistry (i.e., Sortase A (SrtA)-mediated transpeptidation) for dynamic tun- ing hydrogel sti ness and the presence of bioactive ligands. The rst objective was to establish a tunable and cytocompatible enzymatic scheme for softening cell-laden hydrogels. Brie y, the e ects of SrtA-mediated matrix cleavage were investigated us- ing poly(ethylene glycol) (PEG)-peptide hydrogels crosslinked by SrtA-sensitive and insensitive peptides. Initially, the e ects of various parameters with respect to cat- alytic reactions of SrtA were characterized rheologically, including enzyme and sub- strate concentrations, macromer content, peptide composition, and treatment time. Gel moduli pre- and post-enzyme treatment were measured to verify SrtA-mediated hydrogel softening. The cytocompatibility of SrtA-mediated gel softening system was investigated using human mesenchymal stem cell (hMSC). Upon treatment with SrtA and an oligoglycine substrate, encapsulated hMSCs exhibited extensive spreading in comparison to those within statically sti matrices. The second objective was to es- tablish a reversible ligand exchange system utilizing SrtA-mediated transpeptidation. SrtA-sensitive pendant ligands were immobilized within PEG hydrogels, which were treated with SrtA and an oligoglycine substrate to a ord tunable removal of the pen- dant ligand. Through measurement of the liberated pendant peptide concentration, it was found that higher concentrations of SrtA or extending treatment times led to higher ligand removal e ciency. Finally, the e ect of peptide ligand removal on cell behaviors were evaluated using NIH 3T3 broblasts. Fibroblasts were culture both on and within hydrogels containing SrtA-cleavable cell adhesion peptide. After treatment, both conditions led to a decrease in broblast spreading in comparison to non-treated gels. Overall, the utility of SrtA as versatile agent for controlling the mechanical properties and the presence of biologically active components within a hydrogel system was demonstrated. These systems could be further explored with natural-based materials to better mimic the physiological environment experienced by cells.

Final publication is available at http://onlinelibrary.wiley.com/doi/10.1002/term.2060/abstract;jsessionid=F0849D98381EEF9E83401A02B9042F4D.f04t02 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19602号 / 医博第4109号 / 新制||医||1014(附属図書館) / 32638 / 京都大学大学院医学研究科医学専攻 / (主査)教授 別所 和久, 教授 伊佐 正, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13051号 / 論医博第2117号 / 新制||医||1017(附属図書館) / 33141 / (主査)教授 別所 和久, 教授 椛島 健治, 教授 開 祐司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20246号 / 医博第4205号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 杉田 昌彦, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20807号 / 医博第4307号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 瀬原 淳子, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13195号 / 論医博第2159号 / 新制||医||1030(附属図書館) / (主査)教授 松田 秀一, 教授 三森 経世, 教授 妻木 範行 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Equine hyperelastosis cutis (HC or HERDA), a connective tissue disorder in American Quarter Horses, results in hyperelastic skin with poor wound healing. Similar conditions are found in many species and all forms display decreased skin tensile strength. Fibroblasts produce collagen and elastin fibers, forming networks, providing the dermis with strength, and elasticity. This study aims to carry out a 3-part evaluation between horse skin fibroblast (cells from horses affected with HERDA, cells from horses that are carriers of HERDA (recessive HERDA gene), and cells from horses that are normal (neither affected or carriers of HERDA); Studies include: 1. Cell proliferation assay 2. Apoptosis analysis of fibroblasts 3. Mechanobiology of stretched fibroblast. Studies have shown cellular deformation to have an overall effect on mechanical properties of healthy and unhealthy tissues. This investigation provides a micromechanical evaluation of HC/HERDA in an effort to quantify the cellular level differences between each condition.

The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair: the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thymine-containing dimers, and measurement of specific ultraviolet-endonucleasesensitive sites in irradiated DNA. Little effect of hydroxyurea was observed at the concentration of 2 mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65-70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea incubation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well.