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Part I: Characterization of Zn reconstituted Cytochrome B561 Part II: Studies of Catecholamine metabolism in SH-SY5Y and MN9D cells
Hewawitharana, Inoka Samanthi
Hewawitharana, Inoka Samanthi
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Dissertation
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2009-05
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Electronic dissertations
Electronic dissertations
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Abstract
Cytochrome b561, a transmembrane heme protein present in neurotransmitter
storage vesicles, shuttles electrons from the cytosolic Asc to the intragranullar matrix to
regenerate Asc. Although Cyt b561 has been purified, cloned and sequenced from
various sources, the physical structure is yet unresolved. Previous studies have shown
that one of the hemes in the protein is pH labile under mild alkaline conditions. In the
present study, Zn (II) protoporphyrin IX (ZnP) was reconstituted using the altered
protein to further study the mechanism of the transmembrane electron transfer reaction
of Cyt b561. The ZnP reconstituted protein was found to quench 90% of the fluorescence
compared to the same concentration of free ZnP in the solution. The Significance of
these findings with respect to the physiological role of Cyt b561 is discussed.
In part II, the research has focused on two commonly using CNS dopaminergic
models, SH-SY5Y and MN9D cells, which are poorly characterized with respect to their
catecholamine metabolism. Differentiated and undifferentiated cells of both cell lines
were used to detect the baseline levels and uptake studies. After performing the kinetic
experiments for low, medium and high passage cells, we concluded that SH-SY5Y
could not be considered as a fully functional CNS dopaminergic model and medium and
high passage cells may be characterized as noradrenergic. MN9D cells store high
baseline levels of DA but both the differentiated and undifferentiated forms show poor
catecholamine uptake characteristics, probably due to the high intracellular DA levels.
Therefore, MN9D may be a better CNS dopaminergic model with some deficiencies in
DA uptake and release.
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Thesis (Ph.D.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry
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Wichita State University