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.

Thesis (Ph. D.)--University of Iowa, 2008. / Thesis supervisor: Sonya J. Franklin. Includes bibliographical references (leaves 129-134).

No description available.

In the present study non-5-HT₁(A)/non-5-HT₁(C) binding sites in the rabbit caudate nucleus (CN) were examined to determine if they might be identical to the recently discovered 5-HT₁(D) sites in the bovine CN. The characterizations were carried out measuring high-affinity [³H]5-HT binding under conditions where 5-HT₁(A) and 5-HT₁(C) sites were pharmacologically masked in both tissues. Comparison of the pharmacologic profiles of the bovine 5-HT₁(D) and rabbit non-5-HT₁(A)/non-5-HT₁(C) sites revealed similarities, but showed distinct differences. [³H]5-HT binding in the bovine CN was significantly more sensitive to inhibition by GTP than was [³H]5-HT binding in the rabbit CN, and this effect was differentially sensitive to calcium and other divalent cations (i.e., Mg²⁺, Mn²+)⁺in the two tissues. [³H]5-HT binding in the bovine CN was significantly more sensitive to inhibition by NEM than it was in the rabbit CN. Thus, it may be concluded that the non-5-HT₁(A)/non-5-HT₁(C) [³H]5-HT binding sites in rabbit CN are distinct from those in the bovine CN, and we propose that they be tentatively identified as 5-HT₁(R) to distinguish them from the 5-HT₁(D) site.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Thirty six cases of human endometrium (9 normals, 9 adenomatous hyperplasias and 18 cancers) were established as finite monolayer tissue cultures. All were evaluated for the presence of estrogen binding sites (EBS) by an indirect immunofluorescent (IF) technique. Positive EBS were identified in 33% of normals, 67% of hyperplasias and 62% of cancers. Serial subpassage EBS evaluation was performed in fourteen cases. In was observed that the stability of EBS positivity in vitro was related to type of endometrium rather than culture longevity (2 of 2 normals, 2 of 4 hyperplasias and 1 of 8 cancers remained positive throughout the period of study). Twelve of the cancers were studied for estrogen receptor by cytosol assay and 11 (91.6%) correlated with the IF marker method. Thirty-eight cases of breast cancer were studied for EBS by a direct cytochemical and immunofluorescent technique. Evaluation by the direct method proved to be consistent and easy in performance. Morphologic positivity was 60% with the indirect method and 94.7% with the direct method. Correlation with the chemical cytosol was 77% with the indirect method and 86.8% with the direct method. These results confirm and compare favorably with other reported studies of morphologic methods. It was suggested that attention should be directed to cellular localization of receptors as a possible means for predicting endocrine responsiveness of neoplasms. Cancers from tissues presumed to be target-variants for estrogen stimulation were investigated with the direct cytochemical technique to determine if EBS were present. Forty-eight randomly selected tumors from multiple organ systems were assessed for EBS. Appropriate control tests were used to determine specificity. A total of 23 of the 48 cases were interpreted as positive for EBS. These sites were localized to cytoplasmic, nuclear and nucleolar cellular compartments. Estrogen and progesterone receptors in patients with breast carcinoma are of value in the selection of patients for hormonal adjuvant therapy. Whether this will prove to be true for EBS in a variety of neoplasms is currently unknown and is worthy of investigation.

The hydrogenase enzyme is an effective replacement for the expensive platinum catalysts used in hydrogen fuel cells today. However, many enzymes themselves are found in extreme environments and are inactive under standard conditions, but current active site models have a much larger over-potential for H+ reduction than the actual enzyme. Most research today involves the improvement of these synthetic models in an attempt to lower reduction potential, increase reaction kinetics, or improve catalytic activity. Research focuses on the synthesis of active site models with a carbon chain bridgehead linker of varying length. Synthesis of these molecules is achieved by the reaction of a dithiol with triiron dodecacarbonyl under an inert atmosphere to avoid the formation of by-products. Dithiols with four or more carbon atoms must first be converted to cyclic disulfides before the reaction with the iron The hydrogenase enzyme is an effective replacement for the expensive platinum catalysts used in hydrogen fuel cells today. However, many enzymes themselves are found in extreme environments and are inactive under standard conditions, but current active site models have a much larger over-potential for H+ reduction than the actual enzyme. Most research today involves the improvement of these synthetic models in an attempt to lower reduction potential, increase reaction kinetics, or improve catalytic activity. Research focuses on the synthesis of active site models with a carbon chain bridgehead linker of varying length. Synthesis of these molecules is achieved by the reaction of a dithiol with triiron dodecacarbonyl under an inert atmosphere to avoid the formation of by-products. Dithiols with four or more carbon atoms must first be converted to cyclic disulfides before the reaction with the iron dodecacarbonyl. This prevents the formation of an unwanted side product. Both butyl- and pentyldithiolatohexacarbonyldiiron model complexes have been characterized by IR, NMR, and X-ray spectroscopy. Active site models can also feature two unlinked sulfur atoms. These models have two conformational isomers that depend on the spatial location of the R-group bonded to each sulfur atom. This research also focuses on the synthesis of unlinked active site models with a variety of R-groups, and a temperature controlled NMR study of the isomeration reaction to determine the reaction rate. / Review of literature -- Synthesis of [FeFe]-hydrogenase active site mimics with bridged sulfur atoms -- Preliminary kinetics study of [FeFe]-hydrogenase active site mimics. / Department of Chemistry

Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Edward Hawrot. Includes bibliographical references.

published_or_final_version / abstract / toc / Biochemistry / Doctoral / Doctor of Philosophy

Thesis (Ph.D.) --Murdoch University, 2003. / Thesis submitted to the Division of Veterinary and Biomedical Sciences. Includes bibliographical references (leaves 160-196).