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Studies of new factors involved in cholesterol homeostasisDu, Ximing Robin, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2007 (has links)
The regulatory pool of cholesterol is located in the endoplasmic reticulum (ER) and is key to how mammalian cells sense and respond to changes in cellular cholesterol levels. The extent of cholesterol esterification by the ER-resident protein, acyl-coenzyme A:cholesterol acyl-transferase (ACAT), has become the standard method for monitoring cholesterol transport to the ER and is assumed to reflect the regulatory pool of ER cholesterol. The oxysterol, 25-hydroxycholesterol (25HC), is thought to trigger intracellular cholesterol transport to the ER. Chapter 3 of this thesis addressed whether or not cholesterol esterification necessarily reflects cholesterol movement to the cholesterol homeostatic machinery in the ER as determined by SREBP processing. Three agents that inhibited the ability of 25HC to induce cholesterol esterification (progesterone, nigericin, and monensin) did not have a corresponding effect on 25HC suppression of SREBP processing. Moreover, ACAT inhibition did not alter the sensitivity of SREBP processing to 25HC. These findings indicate that cholesterol esterification by the ER-resident protein ACAT is dissociable from cholesterol transport to the cholesterol homeostatic machinery in the ER. In order to readily analyse and quantify the processing of SREBP-2, the master regulator of cholesterol homeostasis, the second part of this study (Chapter 4) established a stable cell-line (13A/PS) that expresses the fusion protein PLAP-BP2 (secreted form of placental alkaline phosphatase joined to the luminal loop of SREBP-2 on the NH2- terminal side of Site-1). Processing of PLAP-BP2 mimics the cleavage of SREBP. The PLAP-BP2 cleavage assay was shown to be a valuable method for analysing and quantifying SREBP-2 processing. Compared to transiently transfected cells, the stable 13A/PS cells were more responsive to changes of sterol status in regards to PLAP secretion. Considering the reliability and consistency of the experiments, the 13A/PS cell-line is therefore a useful tool for the study of SREBP-2 processing. Akt is a critical regulator of cell growth, proliferation and survival that is activated by phosphatidylinositol 3-kinase (PI3K). Chapter 5 and Chapter 6 investigated the role of PI3K/Akt in activation of SREBP-2 increased in response to statin treatment and various cholesterol depletion approaches. The increase of SREBP-2 processing by statins or insulin-like growth factor-1 was blunted by treatment with a potent and specific inhibitor of PI3K, LY294002, or when a plasmid encoding a dominant-negative form of Akt (DNAkt) was expressed. Furthermore, LY294002 treatment down-regulated SREBP-2 or -1c gene targets, and decreased cholesterol and fatty acid synthesis. Fluorescence microscopy studies indicated that LY294002 disrupts transport of the SREBP escort protein, SCAP, from the ER to the Golgi. This disruption was also shown by immunofluorescence staining when DN-Akt was expressed. These data indicate that the PI3K/Akt pathway is involved in SREBP-2 transport to the Golgi, contributing to the control of SREBP-2 activation. The results provide a crucial mechanistic link between the SREBP and PI3K/Akt pathways which may be reconciled teleologically since synthesis of new membrane is an absolute requirement for cell growth and proliferation. This thesis has provided the first evidence that there is an Akt dependent step in SREBP/SCAP transport to the Golgi, adding another input into SREBP activation beside the familiar lipid end products. However, since the Akt protein target involved in the ER-to-Golgi transport of SREBP/SCAP is still elusive, further studies are required to elucidate how Akt exerts its effects, which will potentially reveal new component(s) or pathway(s) involved in cellular cholesterol homeostasis.
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The synthesis of 10a cholesterol.Lawson, Nelson Ernest January 1967 (has links)
No description available.
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Studies of new factors involved in cholesterol homeostasisDu, Ximing Robin, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2007 (has links)
The regulatory pool of cholesterol is located in the endoplasmic reticulum (ER) and is key to how mammalian cells sense and respond to changes in cellular cholesterol levels. The extent of cholesterol esterification by the ER-resident protein, acyl-coenzyme A:cholesterol acyl-transferase (ACAT), has become the standard method for monitoring cholesterol transport to the ER and is assumed to reflect the regulatory pool of ER cholesterol. The oxysterol, 25-hydroxycholesterol (25HC), is thought to trigger intracellular cholesterol transport to the ER. Chapter 3 of this thesis addressed whether or not cholesterol esterification necessarily reflects cholesterol movement to the cholesterol homeostatic machinery in the ER as determined by SREBP processing. Three agents that inhibited the ability of 25HC to induce cholesterol esterification (progesterone, nigericin, and monensin) did not have a corresponding effect on 25HC suppression of SREBP processing. Moreover, ACAT inhibition did not alter the sensitivity of SREBP processing to 25HC. These findings indicate that cholesterol esterification by the ER-resident protein ACAT is dissociable from cholesterol transport to the cholesterol homeostatic machinery in the ER. In order to readily analyse and quantify the processing of SREBP-2, the master regulator of cholesterol homeostasis, the second part of this study (Chapter 4) established a stable cell-line (13A/PS) that expresses the fusion protein PLAP-BP2 (secreted form of placental alkaline phosphatase joined to the luminal loop of SREBP-2 on the NH2- terminal side of Site-1). Processing of PLAP-BP2 mimics the cleavage of SREBP. The PLAP-BP2 cleavage assay was shown to be a valuable method for analysing and quantifying SREBP-2 processing. Compared to transiently transfected cells, the stable 13A/PS cells were more responsive to changes of sterol status in regards to PLAP secretion. Considering the reliability and consistency of the experiments, the 13A/PS cell-line is therefore a useful tool for the study of SREBP-2 processing. Akt is a critical regulator of cell growth, proliferation and survival that is activated by phosphatidylinositol 3-kinase (PI3K). Chapter 5 and Chapter 6 investigated the role of PI3K/Akt in activation of SREBP-2 increased in response to statin treatment and various cholesterol depletion approaches. The increase of SREBP-2 processing by statins or insulin-like growth factor-1 was blunted by treatment with a potent and specific inhibitor of PI3K, LY294002, or when a plasmid encoding a dominant-negative form of Akt (DNAkt) was expressed. Furthermore, LY294002 treatment down-regulated SREBP-2 or -1c gene targets, and decreased cholesterol and fatty acid synthesis. Fluorescence microscopy studies indicated that LY294002 disrupts transport of the SREBP escort protein, SCAP, from the ER to the Golgi. This disruption was also shown by immunofluorescence staining when DN-Akt was expressed. These data indicate that the PI3K/Akt pathway is involved in SREBP-2 transport to the Golgi, contributing to the control of SREBP-2 activation. The results provide a crucial mechanistic link between the SREBP and PI3K/Akt pathways which may be reconciled teleologically since synthesis of new membrane is an absolute requirement for cell growth and proliferation. This thesis has provided the first evidence that there is an Akt dependent step in SREBP/SCAP transport to the Golgi, adding another input into SREBP activation beside the familiar lipid end products. However, since the Akt protein target involved in the ER-to-Golgi transport of SREBP/SCAP is still elusive, further studies are required to elucidate how Akt exerts its effects, which will potentially reveal new component(s) or pathway(s) involved in cellular cholesterol homeostasis.
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Observations on dietary factors affecting serum cholesterolSeidel, John Charles, January 1961 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.
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Microbiological reduction of cholesterol in vitroKellogg, Thomas F. January 1965 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1965. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.
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Purification, properties and regulation of rat liver B-hydroxy-B-methylglutaryl coenzyme A reductase and studies on the coordinate control of lipogenic enzymes in insulinKleinsek, Don Alan. January 1900 (has links)
Thesis--University of Wisconsin--Madison. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.
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Purification and properties of [beta]-hydroxy-[beta]-methylglutaryl coenzyme A reductase of yeast and studies on cholesterol synthesis in hog aorta /Qureshi, Nilofer, January 1975 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1975. / Typescript. Vita. Includes bibliographical references.
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Cholesterol, a physical, chemical and biological study.Huntsinger, Mildred Elizabeth January 1935 (has links)
Thesis (Ph.D.)--Boston University.
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The synthesis of 10a cholesterol.Lawson, Nelson Ernest January 1967 (has links)
No description available.
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A comparison of cholesterol measurements via various blood sample typesMacklin, Diana C. January 1991 (has links)
There has been inconsistencies in the performance of dry-chemistry analyzers using different blood sample types. Therefore, the purpose of this study was to determine precision and accuracy of both capillary and venous whole blood analyzed by the Reflotron (Boehringer Mannheim Diagnostics, Indianapolis, Indiana) and capillary plasma analyzed by the Ektachem DT60 (Eastman Kodak Company, Rochester, New York). Fifty subjects were recruited to provide a representative sample of cholesterol concentrations. One technician performed two fingerstick punctures and one venipuncture on each subject and analyzed the blood sample types in duplicate using each of the dry-chemistry analyzers. The methods and sample types utilized for comparison of total cholesterol are summarized below.ReflotronEktachem DT60Sigma2-Fingerstick whole2-Fingerstick plasma2-Venipuncture plasmablood2-Venipuncture whole2-Venipuncture plasmablood2-Veni uncture lasmaThe mean percent variation of the duplicate samples analyzed revealed all sample types, with the exception of fingerstick whole blood analyzed by theReflotron, met the LSP ideal goal for precision of 5 3% CV. Fingerstick wholeblood CV was 3.1%, meeting the current LSP standard of _5 5% CV for precision. The Sigma wet-chemistry assay for determination of total cholesterol was used as the reference for assessment of bias of each of the sample types. Fingerstick whole blood, via the Reflotron method, produced a positive 5.5% bias when compared to the reference, failing to meet the current LSP goal for acceptable accuracy (±5% bias). Venous whole blood analyzed using the Reflotron met this goal with a bias of +3.3%. Fingerstick plasma, via the Ektachem DT60 method, produced a bias of +2.1%, meeting the ideal LSP goal of ±3% bias. Venous plasma as measured by both the Reflotron and Ektachem DT60 also met this ideal goal (+2.0% and +1.8% bias, respectively). Overall, precision and accuracy of all sample types, with the exception of fingerstick whole blood, when analyzed by their respective dry-chemistry analyzer was acceptable. / School of Physical Education
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