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Browsing by Author "Deluca, Jennifer, committee member"

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    Investigating the osteogenic potential of multipotent mesenchymal stromal cells through the use of DNA microarray technology and biomaterial nanotopography
    (Colorado State University. Libraries, 2011) Berger, Dustin, author; Prasad, Ashok, advisor; Popat, Ketul, advisor; Deluca, Jennifer, committee member
    To view the abstract, please see the full text of the document.
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    Proteomic analysis of the effect of metabolic acidosis on the apical membrane of the renal proximal convoluted tubule
    (Colorado State University. Libraries, 2011) Walmsley, Scott J., author; Curthoys, Norman, advisor; Deluca, Jennifer, committee member; Dobos, Karen, committee member; Laybourne, Paul, committee member; Prenni, Jessica, committee member
    Metabolic acidosis is a physiological disturbance which results in a decrease in blood and extracellular pH and HCO3-. The renal response to this disturbance is initiated in the proximal convoluted tubule (PCT) of the kidney. At the PCT, the brush border membrane facilitates solute reabsorbtion and excretion of acid during acidosis. However, the extent of the global remodeling of proteins at the brush border remains mostly unknown. Therefore a proteomic investigation of the remodeling of theseproteins during metabolic acidosis at the brush border was completed. First, using LTQ mass spectrometry and spectral counting, an enrichment method was tested that analyzed brush border membrane vesicles (BBMV) from cortex versus those which were derived from purified proximal convoluted tubules. From these results we detected and hypothesized that enzymes of glucose metabolism localized at the brush border would be altered in abundance during acidosis at the PCT brush border. Next, we performed a quantitative analysis of the temporal response to metabolic acidosis during 1-d, 3-d and 7-d acidosis using Q-TOF mass spectrometry and spectral counting. As expected, the results indicated a decrease of enzymes of glucose metabolism including Fructose-1,6-bisphosphatase 1 and Enolase A. Aldolase A was found to be transiently decreased during 1-d and 3-d acidosis. In addition, the Na+-glucose transporter 2 was found to be transiently increased during 1-d and 3-d acidosis. Finally, to confirm these abundance changes detected using spectral counting, an accurate mass and time tag method was developed. Using this method, we successfully developed an AMT database of the previously identified spectra. This database was used to match peptides detected using QTOF-LC-MS to the previously identified peptides. Peptide abundance by spectral counting was validated using the more accurate peak intensities and were generally in concordance with those abundance measurements using spectral counting. The developed model suggested a mechanism for internalization of these enzymes of glucose metabolism in support of glutamine metabolism, which is central to the cellular response to acidosis by the PCT.
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    Structural and functional insight into kinetochore protein CENP-N and its interaction with CENP-A nucleosome
    (Colorado State University. Libraries, 2018) Zhou, Keda, author; Luger, Karolin, advisor; Yao, Tingting, committee member; Deluca, Jennifer, committee member; Bailey, Susan, committee member
    Proper chromosome segregation during mitosis is one of the most important processes to ensure genome integrity. During this process, the microtubules are captured by a multi-unit complex called kinetochore. The kinetochore is assembled specifically at centromere through recognizing nucleosomes containing the histone H3 variant CENP-A. CENP-N and CENP-C are the only two kinetochore proteins that specifically recognize CENP-A nucleosomes. There are about 1 in 25 nucleosomes that contain CENP-A at the centromere. Therefore, how these two proteins 'ignore' the abundant H3 nucleosomes to interact selectively with a handful of centromeric CENP-A nucleosomes has important implications for genome stability during cell division. To obtain deep insight into the mechanism behind this, I solved the structure of CENP-A nucleosome in complex with CENP-N by single particle cryo electron microscopy (cryo-EM) at 4 Å. Through charge and space complementarity, the unique "RG" loop on CENP-A is decoded by CENP-N. CENP-N also engages in extensive interactions with a long segment of the distorted nucleosomal DNA double helix. These interactions were validated in vitro and in vivo.The DNA ends of CENP-A nucleosome which are disordered in the crystal structure are mostly visible in the cryo-EM structure when it is in complex with CENP-N. By micrococcal nuclease digestion assay, the CENP-A nucleosome DNA ends are shown to be less flexible when CENP-N is presented in solution, which is consistent with structural study. Since CENP-N does not interact with DNA ends directly, the less dynamics on the DNA ends indicate a more stable nucleosome. By quantitative electrophoretic mobility shift assay (EMSA) and electron microscopy, the stabilizing effect of CENP-N on CENP-A nucleosome was confirmed in vitro. However, this effect was not significant in vivo, which indicates that the CENP-A nucleosome stability in vivo is determined by multiple factors. Besides the change on DNA ends of CENP-A nucleosome, the orientation of H4 N-terminal tail is altered due to its interaction with CENP-N, with important implications for the multiple biological processes involving the H4 N-terminal tail, especially with respect to the formation of chromatin higher order structure The structural and functional studies in this thesis shed light on how CENP-N ensures that the kinetochore assembles specifically at the centromere.