The centromere binding factor 1, Cbf1, of Saccharomyces cerevisiae is a bHLH/ZIP protein which has been described as a determinant of specific chromatin structures and as a tethering factor for activators of transcription at the promoters of genes of the Methionine Biosynthesis Pathway. Deletion mutants show various phenotypes, among them methionine auxotrophy, an increased rate of chromosome loss, modifications in the growth rate and modification of the chromatin structure at MET genes. Meiosis competence also becomes greatly reduced in cbf1 cells. The sequence motif (RTCACRTG) to which Cbf1p binds is found at multiple loci through the yeast genome. This thesis shows that the chromatin structure is reorganised at multiple Cbf1p binding sites in vivo, when yeast cells are starved to enter meiosis. Extensive remodelling occurs at the MET16, MET17(25), DRS2 and GDH3 loci and at the YAL060W open reading frame, as detected by in vivo digestion of chromatin with micrococcal nuclease and indirect end-labelling. The same kind of analysis showed that the remodelling of chromatin at Cbf1p binding sites is not specific for meiosis, it occurs also in similarly starved haploid cells. The lack of methionine is a key trigger of these changes. This reorganisation of chromatin is dependent on Cbf1p, since starved cbf1 cells do not display any modification in nuclease accessibility patterns at or around Cbf1p binding sites. Mutational analysis revealed that a negative charge at a putative phosphorylation site (serine residue 226) and the DNA-bindmg activity of Cbf1p are both required for the chromatin reorganisation to occur in response to starvation. CBF1 mutants which do not reorganise chromatin were also shown to be unable to enter meiosis, suggesting that the remodelling of chromatin at multiple Cbf1p binding sites may be required to enter pre-meiotic DNA replication, since such cells arrest before the initiation of this process. In summary, the results presented in this thesis are compatible with a model in which Cbf1p plays an active role as part of a mechanism sensing the nutrient availability and regulates the reorganisation of chromatin, at multiple loci through the yeast genome, in response to starvation conditions.

No description available.

No description available.

No description available.

The multigene family of Ca2+-binding S100 proteins comprises 22 members that have various important intra- and extracellular roles. The three inflammation-associated members of this family???S100A8, S100A9 and S100A12 (collectively termed &quotcalgranulins&quot)???are constitutive neutrophil and monocyte proteins also expressed by macrophages within acute and chronic inflammatory lesions, but not in tissue macrophages. They are expressed in human/murine wounds and by appropriately activated macrophages, microvascular endothelial cells and keratinocytes in vitro. The &quot calgranulins&quot are implicated in leukocyte activation/deactivation, fatty acid transport, leukocyte/fibroblast chemotaxis, transmigration and adhesion, embryogenesis, wound healing, protection against oxidants and antibacterial defence. Chapter 3 of this thesis explores growth-factor- and cytokine-mediated regulation and expression of S100A8 and S100A9 in fibroblasts, and demonstrates spatio-temporal expression of S100A8 in rat dermal wounds. Fibroblasts are stromal resident cells with important regulatory immune-inflammatory functions in wound healing, tissue remodelling and fibrosis. Fibroblast migration, proliferation, differentiation and their synthetic repertoire are modulated by various factors including extracellular matrix components, growth factors, prostaglandins, reactive oxygen species and cytokines. Fibroblast growth factor-2 (FGF-2), interleukin-1?(IL-1? and platelet-derived growth factor (PDGF) are potent fibroblast mitogens; PDGF and transforming growth factor-? (TGF-? are fibroblast chemoattractants. FGF-2 and IL-1?promote fibroblast proliferation, whereas TGF-?promotes myofibroblast differentiation and collagen production. Lipopolysaccharide (LPS), interferon ?(IFN?, tumour-necrosis factor ? (TNF?, TGF-?and PDGF did not induce the S100A8 gene in fibroblasts whereas FGF-2 (25 ng/ml) maximally induced mRNA 12 hr. after stimulation and this declined over 36 hr. The FGF-2 response was strongly enhanced and prolonged by optimal levels of heparin (1-10 IU/ml), maximally at 18 hr. post-stimulation. FGF-2/heparin-induced responses depended on cell-cell contact in vitro. IL-1?(10 U/ml) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 and primary fibroblasts. Dexamethasone (10???6 M) enhanced LPS- and FGF-2/-IL-1?induced responses. S100A9 mRNA was not induced by any of these mediators. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblast-like cells by real-time reverse-transcriptase polymerase chain-reaction. FGF-2-heparin- and IL-1?induced mRNA expression depended on de-novo protein synthesis and was partially mediated by the mitogenactivated protein kinase pathway of activation. Preliminary promoter deletion analyses indicated that FGF-2-responsive elements in the gene promoter were distinct from those responsive to IL-1? TGF-?(2 ng/ml) significantly suppressed gene induction mediated by FGF-2 ?heparin/LPS/dexamethasone, but not by IL-1? TGF-?may compromise mRNA stability. Protein levels in FGF-2-heparin-IL-1?stimulated fibroblasts correlated well with mRNA levels and expression was mainly cytoplasmic. Immunohistochemistry indicated S100A8 associated with keratinocytes, neutrophils, macrophage-like cells and some hair follicles in wounded rat skin. Rat wounds also contained numerous S100A8- positive fibroblast-like cells 2 and 4 days post-injury; numbers declined by 7 days. Upregulation of S100A8 by FGF-2/IL-1? down-regulation by TGF-? and time-dependent expression of S100A8 in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair. Intracellular fibroblast-derived S100A8 may also regulate intracellular redox equilibrium and antioxidant defence. Atherosclerosis is a progressive chronic disease with complex aetiology and pathogenesis. S100A1 and S100B are associated with dendritic cells and lymphocytes in experimental rodent and human atherosclerotic lesions. Monocytes and macrophages in plaques of ApoE???/??? mice express S100A9 but not S100A8. Myeloperoxidase and HOClmediated oxidative mechanisms are fundamental in the pathogenesis of atherosclerosis and S100A8 is exquisitely sensitive to HOCl oxidation which generates sulphinamide bonds, novel non-reducible cysteine-lysine covalent bonds. Chapter 4 of this thesis presents novel evidence that, in contrast to the murine ApoE???/??? model, the three human &quot calgranulins&quot were expressed in human atherosclerotic plaques, but not in normal arteries. High levels of S100A8, S100A9 and S100A12 were evident in macrophages and foam cells. Some neovessels were anti-S100A8-/anti-S100A9-immunoreactive; S100A9 staining was also evident on the extracellular matrix. Patterns of expression of S100A8, S100A9 and S100A12 were overlapping in serial sections, except that only smooth muscle cells were S100A12-positive. S100A8 and S100A9 mRNA were also expressed by macrophages, foam cells and endothelial cells, indicating gene up-regulation rather than passive protein uptake. Western blotting of plaque extracts revealed monomeric S100A8, S100A9 and S100A12 and larger complexes. Some were resistant to reduction, suggesting non-disulfide covalent cross-linking, possibly via sulphinamide bonds. Stable S100A8-S100A9 complexes were also detected after immunoaffinity purification. In an in-vitro system, molar ratios of HOCl of &gt1 generated stable complexes of S100A8 and S100A9 whereas ~800 and ~100-fold excess HOCl oxidises apolipoprotein B-100 and BSA, respectively. S100A8 and S100A9 protected low-density lipoprotein (LDL) against HOCl oxidation in a thiol-independent manner. Because HOCl-oxidised S100s did not contain epitopes recognised by an antibody used to detect HOCl-oxidised proteins in plaque, levels of oxidised proteins in plaque are likely to be significantly greater than described. S100A8 and S100A9 may protect LDL by functioning as HOCl-scavengers. However, chronic oxidative cross-linking of S100A8 and S100A9 with other proteins and extracellular matrix components may contribute to plaque pathogenesis. These studies support key roles for the &quot calgranulins&quot in chronic inflammation, wound healing and atherogenesis possibly by regulating cellular differentiation, activation and modulation of redox-dependent mechanisms.

Thesis (Ph. D.)--University of North Texas, Aug., 2007. / Title from title page display. Includes bibliographical references.

Thesis (Ph. D.)--University of New South Wales, 2004. / Also available online.

Thesis (Ph.D.)--Murdoch University, 2006. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 257-284.

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains ix, 65 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 58-62).

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: Role of Tryptophan in cytochrome b₅. Includes bibliographical references (187-196). Also available online through Digital Dissertations.