+publisher:"Colorado State University" +contributor:("Curthoys, Norman P.")

Colorado State University

1. Schauer, Kevin Lee. Temporal changes in the cytosolic proteome of the proximal convoluted tubule during the onset of metabolic acidosis.

Degree: MS(M.S.), Biochemistry and Molecular Biology, 2013, Colorado State University

URL: http://hdl.handle.net/10217/79164

► A decrease in blood pH coupled with a decrease in blood bicarbonate concentration is a relatively common pathological condition that is referred to as metabolic… (more)

Subjects/Keywords: metabolic acidosis; spectral counting; proximal convoluted tubule; proteomics

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APA (6^th Edition):

Schauer, K. L. (2013). Temporal changes in the cytosolic proteome of the proximal convoluted tubule during the onset of metabolic acidosis. (Masters Thesis). Colorado State University. Retrieved from http://hdl.handle.net/10217/79164

Chicago Manual of Style (16^th Edition):

Schauer, Kevin Lee. “Temporal changes in the cytosolic proteome of the proximal convoluted tubule during the onset of metabolic acidosis.” 2013. Masters Thesis, Colorado State University. Accessed April 17, 2021. http://hdl.handle.net/10217/79164.

MLA Handbook (7^th Edition):

Schauer, Kevin Lee. “Temporal changes in the cytosolic proteome of the proximal convoluted tubule during the onset of metabolic acidosis.” 2013. Web. 17 Apr 2021.

Vancouver:

Schauer KL. Temporal changes in the cytosolic proteome of the proximal convoluted tubule during the onset of metabolic acidosis. [Internet] [Masters thesis]. Colorado State University; 2013. [cited 2021 Apr 17]. Available from: http://hdl.handle.net/10217/79164.

Council of Science Editors:

Schauer KL. Temporal changes in the cytosolic proteome of the proximal convoluted tubule during the onset of metabolic acidosis. [Masters Thesis]. Colorado State University; 2013. Available from: http://hdl.handle.net/10217/79164

Colorado State University

2. Mudron, Megan Reese. Molecular regulation of growth and molting in decapod crustaceans.

Degree: MS(M.S.), Biology, 2014, Colorado State University

URL: http://hdl.handle.net/10217/84008

► The green shore crab, Carcinus maenas, is a highly invasive species that inhabits coastal temperate zones worldwide. The reaction of C. maenas to acute temperature… (more)

▼ The green shore crab, Carcinus maenas, is a highly invasive species that inhabits coastal temperate zones worldwide. The reaction of C. maenas to acute temperature change was determined in six tissues (heart, gill, thoracic ganglion, eyestalk ganglion, Y-organ, and claw muscle) using genetic markers for temperature-induced metabolic stress, including HSP70, AMPKγ, mTOR, and Rheb. Animals were exposed to temperatures between 5° and 30°C for 1 or 2 h. mRNA levels in six tissues were quantified by quantitative RT-PCR (qPCR). The results indicate that C. maenas tolerated a wide temperature range, requiring 2-h exposures at 5 °C and 30 °C to affect tissue-specific changes in gene expression. Cm-HSP70 expression was robustly increased at 30 °C in all tissues. Ecdysteroids produced from the molting gland (Y-organ or YO) induce molting in decapod crustaceans. Reduction in molt-inhibiting hormone (MIH) activates the YO and animals enter premolt. At mid-premolt, YOs transition to the committed state, during which ecdysteroid production increases further. In blackback land crab (Gecarcinus lateralis), a tropical decapod species, SB1431542, an inhibitor of Activin receptors, decreases hemolymph ecdysteroid titers in premolt animals, suggesting that an Activin-like transforming-growth factor (TGF-β) is produced by the activated YO and drives the transition of the YO to the committed state. Myostatin (Gl-Mstn) is an Activin-like factor that is highly expressed in skeletal muscle. Rapamycin lowers hemolymph ecdysteroid titers by inhibiting mTOR, which controls global translation of mRNA into protein. Endpoint RT-PCR established that Gl-Mstn was expressed in the YO, not just muscle tissue. YOs were harvested from intact (intermolt) animals and from animals at 1, 3, 5, 7, and 14 days post-ESA. Quantitative PCR was used to quantify the effects of molt induction by eyestalk ablation (ESA) on gene expression. Expression of mTOR components peaked at 3 days post-ESA, which is consistent with the increased activity required for activation of the YO. Gl-Mstn expression also peaked at 3 days post-ESA, which is before the transition to the committed state at 7 days post-ESA. These results indicate that mTOR components are involved in activation of the YO, and Mstn is involved in transitioning the YO to the committed state. Advisors/Committee Members: Mykles, Donald L. (advisor), Garrity, Deborah M. (committee member), Curthoys, Norman P. (committee member).

Subjects/Keywords: AMPK; crustacean; HSP70; molting; mTOR; myostatin

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Mudron, M. R. (2014). Molecular regulation of growth and molting in decapod crustaceans. (Masters Thesis). Colorado State University. Retrieved from http://hdl.handle.net/10217/84008

Chicago Manual of Style (16^th Edition):

Mudron, Megan Reese. “Molecular regulation of growth and molting in decapod crustaceans.” 2014. Masters Thesis, Colorado State University. Accessed April 17, 2021. http://hdl.handle.net/10217/84008.

MLA Handbook (7^th Edition):

Mudron, Megan Reese. “Molecular regulation of growth and molting in decapod crustaceans.” 2014. Web. 17 Apr 2021.

Vancouver:

Mudron MR. Molecular regulation of growth and molting in decapod crustaceans. [Internet] [Masters thesis]. Colorado State University; 2014. [cited 2021 Apr 17]. Available from: http://hdl.handle.net/10217/84008.

Council of Science Editors:

Mudron MR. Molecular regulation of growth and molting in decapod crustaceans. [Masters Thesis]. Colorado State University; 2014. Available from: http://hdl.handle.net/10217/84008

Colorado State University

3. Angala, Shiva kumar. Mycobacterial arabinan biosynthesis: characterization of AftB and AftC arabinosyltransferase activity using synthetic substrates.

Degree: PhD, Microbiology, Immunology, and Pathology, 2011, Colorado State University

URL: http://hdl.handle.net/10217/70436

► Tuberculosis (TB) is a chronic infectious disease caused by M. tuberculosis (Mtb). Treatment of TB is prolonged, and multidrug resistant (MDR-TB) and extensively drug resistant… (more)

▼ Tuberculosis (TB) is a chronic infectious disease caused by M. tuberculosis (Mtb). Treatment of TB is prolonged, and multidrug resistant (MDR-TB) and extensively drug resistant TB (XDR-TB) cases are ever increasing. Efforts to discover and develop new drugs have increased in recent years so improvement of the existing therapies is urgently needed. The cell wall of Mtb with its unique physiological properties has historically been an important and valid drug target. Mycobacterial arabinosyltransferases are membrane bound glycosyltransferases involved in the biosynthesis of the arabinan portion of two major polysaccharides, arabinogalactan (AG) and lipoarabinomannan (LAM), associated with the cell wall. In this work, M. smegmatis was used as a model organism to study arabinosyltransferases and the biosynthesis of cell wall arabinofuran—the main constituent of AG and LAM. This dissertation addresses the development of a cell free arabinosyltransferase assay for AftB and AftC glycosyltransferases. Since it was not possible to express AftB transmembrane protein, we probed AftB transferase activity from the crude membranes using a synthetic arabinose disaccharide acceptor. In this study, a robust cell free iii radioactive arabinosyltransferase assay was developed using a linkage specific synthetic disaccharide acceptor. Relative mobility of the enzymatic product on a thin layer chromatogram and autoradiography clearly demonstrated the enzymatic conversion of the disaccharide acceptor to a trisaccharide. The trisaccharide product was further confirmed by matrix assisted laser desorption ionization- time of flight (MALDI-TOF) or high pH anion exchange chromatography (HPAEC) analysis. GC-MS analysis showed that the additional arabinose added to the enzymatic product was (1→2) linked. This assay is dependent on time and enzyme concentration and the product formation is not sensitive to the action of ethambutol or the absence of the putative arabinosyltransferases encoded by embA, embB or embC. Additionally, further optimal conditions were determined including buffers, pH, divalent cations, detergents, and the effect of alkylating and reducing agents. In a contiguous study, we wanted to convert this assay into a non-radioactive assay for the screening of compounds. We successfully developed an ELISA based non-radiolabeled arabinosyltransferase assay using CS35-mAb that recognized (1→2) linked enzymatic product. As this method involves several washing steps and time consuming processes, we further modified the substrate with fluorescent labeling, however, we failed to establish a fluorescence based arabinosyltransferase assay. Unlike AftB, in a parallel study we were successful in expressing, solubilizing and purifying Mtb AftC. We developed an in vitro transferase assay using purified recombinant AftC and demonstrated that AftC retains transferase activity only when reconstituted into proteoliposomes. Additionally, we were successful in synthesizing alternate arabinose donors Z-nerylphosphoryl D-arabinose (Z-NPA),… Advisors/Committee Members: Chatterjee, Delphi (advisor), Crick, Dean C. (advisor), Schweizer, Herbert P. (committee member), Curthoys, Norman P. (committee member).

Subjects/Keywords: arabinose; mycobacteria; arabinosyltransferase

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Angala, S. k. (2011). Mycobacterial arabinan biosynthesis: characterization of AftB and AftC arabinosyltransferase activity using synthetic substrates. (Doctoral Dissertation). Colorado State University. Retrieved from http://hdl.handle.net/10217/70436

Chicago Manual of Style (16^th Edition):

Angala, Shiva kumar. “Mycobacterial arabinan biosynthesis: characterization of AftB and AftC arabinosyltransferase activity using synthetic substrates.” 2011. Doctoral Dissertation, Colorado State University. Accessed April 17, 2021. http://hdl.handle.net/10217/70436.

MLA Handbook (7^th Edition):

Angala, Shiva kumar. “Mycobacterial arabinan biosynthesis: characterization of AftB and AftC arabinosyltransferase activity using synthetic substrates.” 2011. Web. 17 Apr 2021.

Vancouver:

Angala Sk. Mycobacterial arabinan biosynthesis: characterization of AftB and AftC arabinosyltransferase activity using synthetic substrates. [Internet] [Doctoral dissertation]. Colorado State University; 2011. [cited 2021 Apr 17]. Available from: http://hdl.handle.net/10217/70436.

Council of Science Editors:

Angala Sk. Mycobacterial arabinan biosynthesis: characterization of AftB and AftC arabinosyltransferase activity using synthetic substrates. [Doctoral Dissertation]. Colorado State University; 2011. Available from: http://hdl.handle.net/10217/70436

Colorado State University

4. Abuhagr, Ali Moftah M. Role of mechanistic Target of Rapamycin (mTOR) signaling in the crustacean molting gland.

Degree: PhD, Biology, 2012, Colorado State University

URL: http://hdl.handle.net/10217/71664

► Regulation of the molt cycle in decapod crustaceans is mainly controlled by the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). Molt-inhibiting hormone (MIH), secreted… (more)

▼ Regulation of the molt cycle in decapod crustaceans is mainly controlled by the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). Molt-inhibiting hormone (MIH), secreted by the XO/SG complex, suppresses production of molting hormone (ecdysteroids) by a pair of YOs. In the blackback land crab, Gecarcinus lateralis, molting can be induced by eyestalk ablation (ESA) or autotomy of 5 or more walking legs (multiple leg autotomy or MLA). During the molt cycle, the YO transitions through four physiological states: "basal" state at postmolt and intermolt; "activated" state at early premolt; "committed" state at mid premolt and "repressed" state at late premolt. The basal to activated state transition is triggered by a transient reduction in MIH; the YOs hypertrophy, but remain sensitive to MIH. The main hypothesis is that up-regulation of mechanistic Target of Rapamycin (mTOR) signaling, which controls global translation of mRNA into protein, is necessary for YO hypertrophy and ecdysteroidogenesis. cDNAs encoding mTOR, Rheb, Akt (protein kinase B) and p70 S6 kinase (S6k) were cloned from blackback land crab, G. lateralis, and green shore crab, Carcinus maenas. All four genes were expressed in all tissues examined. mTOR appears to be involved in YO activation in early premolt, as rapamycin inhibited YO ecdysteroidogenesis in vivo and in vitro. In addition, the expression of Gl-elongation factor 2 (EF2), Gl-mTOR, and Gl-Akt increased significantly in YOs from premolt, suggesting that an increase in protein synthetic capacity is necessary for YO activation. A putative transforming growth factor-beta (TGFâ) appeared to be involved in the transition of the YO from the activated to committed state, as SB431542, an Activin receptor antagonist, lowered hemolymph ecdysteroid titers in mid premolt animals and abrogated the premolt increases in Gl-EF2, Gl-mTOR, and Gl-Akt mRNA levels. By contrast, molting had no effect on Cm-EF2, Cm-mTOR, Cm-Rheb, Cm-Akt, and Cm-S6k expression in C. maenas YOs. Unlike G. lateralis, adult C. maenas was refractory to ESA. ESA caused a small increase in hemolymph ecdysteroid titers, but animals did not immediately enter premolt. Some ES-ablated animals molted after many months, but most failed to molt at all. We hypothesized that other regions of the nervous system, specifically the brain and/or thoracic ganglion, were secondary source(s) of MIH. Nested endpoint RT-PCR showed that MIH transcript was present in brain and thoracic ganglion of intermolt crabs. Sequencing of the PCR product confirmed its identity as MIH. Real time PCR was used to quantify the effects of ESA on MIH expression in brain and thoracic ganglion on C. maenas red and green color morphs. ESA had little effect on MIH transcript levels, indicating that MIH was not regulated transcriptionally by the loss of the eyestalks. The data suggest that MIH secreted by neurons in the brain and thoracic ganglion is sufficient to prevent molt induction when the primary source of MIH is removed by ESA. There was also no effect of ESA on the… Advisors/Committee Members: Mykles, Donald L. (advisor), Garrity, Deborah M. (committee member), Reddy, Anireddy N. (committee member), Curthoys, Norman P. (committee member).

Subjects/Keywords: molting cycle in crustaceans; protein synthesis; mTOR signaling; molting in decapod crustaceans

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Abuhagr, A. M. M. (2012). Role of mechanistic Target of Rapamycin (mTOR) signaling in the crustacean molting gland. (Doctoral Dissertation). Colorado State University. Retrieved from http://hdl.handle.net/10217/71664

Chicago Manual of Style (16^th Edition):

Abuhagr, Ali Moftah M. “Role of mechanistic Target of Rapamycin (mTOR) signaling in the crustacean molting gland.” 2012. Doctoral Dissertation, Colorado State University. Accessed April 17, 2021. http://hdl.handle.net/10217/71664.

MLA Handbook (7^th Edition):

Abuhagr, Ali Moftah M. “Role of mechanistic Target of Rapamycin (mTOR) signaling in the crustacean molting gland.” 2012. Web. 17 Apr 2021.

Vancouver:

Abuhagr AMM. Role of mechanistic Target of Rapamycin (mTOR) signaling in the crustacean molting gland. [Internet] [Doctoral dissertation]. Colorado State University; 2012. [cited 2021 Apr 17]. Available from: http://hdl.handle.net/10217/71664.

Council of Science Editors:

Abuhagr AMM. Role of mechanistic Target of Rapamycin (mTOR) signaling in the crustacean molting gland. [Doctoral Dissertation]. Colorado State University; 2012. Available from: http://hdl.handle.net/10217/71664

Colorado State University

5. Obregón-Henao, Andrés. Novel extracellular products of Mycobacterium tuberculosis: composition, synthesis, and relevance to disease.

Degree: PhD, Microbiology, Immunology, and Pathology, 2013, Colorado State University

URL: http://hdl.handle.net/10217/78863

► Mycobacterium tuberculosis (Mtb) is a bacterium causing great morbidity and mortality especially in developing countries. In order to identify possible areas of intervention to positively… (more)

▼ Mycobacterium tuberculosis (Mtb) is a bacterium causing great morbidity and mortality especially in developing countries. In order to identify possible areas of intervention to positively alter the history of the disease, a better identification and characterization of Mtb virulence determinants is required. Specifically, biosynthetic routes for these virulence determinants should be pursued. Furthermore, the interaction between the host and Mtb virulence determinants should be characterized at a molecular level. It is hoped that unraveling these pathogenesis mechanisms could lead to novel strategies to combat the infection. In Chapter II, the identification of secreted Mtb molecules that induce macrophage apoptosis was performed. Apoptosis is a mechanism of host cell death and in the life cycle of Mtb, different modalities of host cell death have been suggested to tip the balance between bacterial eradication and multiplication. However, a systematic approach to identify and characterize secreted Mtb molecules that modulate host cell death, has not been performed. Surprisingly, extracellular Mtb RNA fragments were identified as a potent inducer of host cell apoptosis. This extracellular RNA was identified as predominantly rRNA and tRNA fragments that accumulated early during in vitro culture of Mtb. Mechanistic studies determined that the Mtb RNA induced macrophage apoptosis through a caspase-8-dependent, TNF-α-independent mechanism. Importantly, Mtb RNA abrogated the macrophage's ability to control an Mtb infection. In Chapter II, the first description of an extracellular Mtb RNA with potent biological activity was performed. This opens an exciting field in research of host interactions with pathogen nucleic acids. Chapters III and IV were devoted to identifying the biochemical pathway involved in α-L-polyGlutamine (α-L-polyGln) biosynthesis and determining its role in pathogenesis in the murine model of TB. α-L-polyGln is an Mtband Mycobacterium bovis (M. bovis) specific product and its presence in virulent Mycobacterium spp., suggest that it could play an important role in pathogenesis. Bacillus anthracis (B. anthracis) synthesizes γ-D-polyGlutamate (γ-D-polyGlu), an amino acid polymer that is present in its capsule and is absolutely required for pathogenicity. As the pathway for B. anthracis γ-D-polyGlu biosynthesis has been well characterized, it was used as a model to start elucidating the Mtb α-L-polyGln biosynthetic pathway. Bioinformatics analysis suggested that Rv0574c and Rv2394 are the Mtb homologues for B. anthracis CapA and CapD, respectively. In Chapter III, a complete biochemical characterization of Rv2394 was performed. Similar to other γ-glutamyltranspeptidases (GGTs), Rv2394 had a conserved catalytic motif consisting of a Threonine (Thr) residue. Mutating this Thr residue to Alanine (Ala) abrogated the enzymatic activity of Rv2394, including its autocatalytic activation. In contrast to eukaryote GGT, Rv2394 was able to perform a GGT activity… Advisors/Committee Members: Belisle, John T. (advisor), Brennan, Patrick J. (committee member), Curthoys, Norman P. (committee member), Dow, Steven W. (committee member).

Subjects/Keywords: latency; poly-glutamine; secretion; transpeptidase; tuberculosis; RNA

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Obregón-Henao, A. (2013). Novel extracellular products of Mycobacterium tuberculosis: composition, synthesis, and relevance to disease. (Doctoral Dissertation). Colorado State University. Retrieved from http://hdl.handle.net/10217/78863

Chicago Manual of Style (16^th Edition):

Obregón-Henao, Andrés. “Novel extracellular products of Mycobacterium tuberculosis: composition, synthesis, and relevance to disease.” 2013. Doctoral Dissertation, Colorado State University. Accessed April 17, 2021. http://hdl.handle.net/10217/78863.

MLA Handbook (7^th Edition):

Obregón-Henao, Andrés. “Novel extracellular products of Mycobacterium tuberculosis: composition, synthesis, and relevance to disease.” 2013. Web. 17 Apr 2021.

Vancouver:

Obregón-Henao A. Novel extracellular products of Mycobacterium tuberculosis: composition, synthesis, and relevance to disease. [Internet] [Doctoral dissertation]. Colorado State University; 2013. [cited 2021 Apr 17]. Available from: http://hdl.handle.net/10217/78863.

Council of Science Editors:

Obregón-Henao A. Novel extracellular products of Mycobacterium tuberculosis: composition, synthesis, and relevance to disease. [Doctoral Dissertation]. Colorado State University; 2013. Available from: http://hdl.handle.net/10217/78863

Colorado State University

6. Lee, Jerome Edward. Global analysis of mRNA decay rates and RNA-binding specificity reveals novel roles for CUGBP1 and PARN deadenylase in muscle cells.

Degree: PhD, Cell and Molecular Biology, 2011, Colorado State University

URL: http://hdl.handle.net/10217/46378

► Type I Myotonic Dystrophy (DM1) is characterized by myotonia, cardiac conduction defects, muscle wasting, and insulin resistance. In patient muscle cells expression and function of… (more)

▼ Type I Myotonic Dystrophy (DM1) is characterized by myotonia, cardiac conduction defects, muscle wasting, and insulin resistance. In patient muscle cells expression and function of the RNA-binding proteins CUGBP1 and MBNL1 are disrupted, resulting in altered mRNA metabolism at the levels of splicing and translation. Intriguingly, despite strong evidence for CUGBP1 being a regulator of mRNA turnover in humans and other organisms, the possibility that defects in mRNA decay contribute to DM1 pathogenesis has not been investigated to date. As such, we sought to further characterize the roles of CUGBP1 and its partner, the deadenylase PARN, in mRNA decay in mouse C2C12 muscle cells. The TNF message, which encodes a cytokine known to cause muscle wasting and insulin resistance when over-expressed, was stabilized by depletion of CUGBP1. The normally rapid decay of the TNF mRNA was also disrupted in cells treated with phorbol ester and this coincided with phosphorylation of CUGBP1. These findings provided impetus to undertake a global analysis of mRNA decay rates in muscle cells. Our investigation revealed that GU- and AU-rich sequence elements are enriched in labile transcripts, which encode cell cycle regulators, transcription factors, and RNA-processing proteins. Transcripts specifically bound to CUGBP1 in myoblasts are linked with processes such as mRNA metabolism, protein targeting to the endoplasmic reticulum, cytoskeletal organization, and transcriptional regulation, all of which have implications for muscle cell biology. Consistent with this, CUGBP1 depletion profoundly altered the formation of myotubes during differentiation. Finally we investigated whether PARN, which interacts with CUGBP1 and mediates rapid deadenylation of TNF in HeLa cell extracts, also plays a role in mediating mRNA decay in muscle. We identified 64 mRNA targets whose decay was dependent on PARN. Moreover, deadenylation of the Brf2 mRNA was impaired in PARN knock-down cells supporting that this mRNA is directly and specifically targeted for decay by PARN. Taken together our findings demonstrate that CUGBP1 and PARN are critical regulators of decay for specific sets of transcripts in muscle cells. It seems likely that some or all of the CUGBP1 targets we have identified may be affected in myotonic dystrophy. Defective mRNA turnover could be linked with defects in myogenesis, TNF over-expression, muscle wasting and/or ER stress, all of which have been documented in DM1. Advisors/Committee Members: Wilusz, Carol J. (advisor), Wilusz, Jeffrey (advisor), Garrity, Deborah M. (committee member), Curthoys, Norman P. (committee member).

Subjects/Keywords: CELF1; muscle; mRNA decay; CUGBP1; gene expression; PARN

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APA (6^th Edition):

Lee, J. E. (2011). Global analysis of mRNA decay rates and RNA-binding specificity reveals novel roles for CUGBP1 and PARN deadenylase in muscle cells. (Doctoral Dissertation). Colorado State University. Retrieved from http://hdl.handle.net/10217/46378

Chicago Manual of Style (16^th Edition):

Lee, Jerome Edward. “Global analysis of mRNA decay rates and RNA-binding specificity reveals novel roles for CUGBP1 and PARN deadenylase in muscle cells.” 2011. Doctoral Dissertation, Colorado State University. Accessed April 17, 2021. http://hdl.handle.net/10217/46378.

MLA Handbook (7^th Edition):

Lee, Jerome Edward. “Global analysis of mRNA decay rates and RNA-binding specificity reveals novel roles for CUGBP1 and PARN deadenylase in muscle cells.” 2011. Web. 17 Apr 2021.

Vancouver:

Lee JE. Global analysis of mRNA decay rates and RNA-binding specificity reveals novel roles for CUGBP1 and PARN deadenylase in muscle cells. [Internet] [Doctoral dissertation]. Colorado State University; 2011. [cited 2021 Apr 17]. Available from: http://hdl.handle.net/10217/46378.

Council of Science Editors:

Lee JE. Global analysis of mRNA decay rates and RNA-binding specificity reveals novel roles for CUGBP1 and PARN deadenylase in muscle cells. [Doctoral Dissertation]. Colorado State University; 2011. Available from: http://hdl.handle.net/10217/46378

Colorado State University

7. Gummadi, Lakshmi. Role of HuR, AUF1 and zeta-crystallin in mediating pH-responsive increase in renal phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance in kidney cells.

Degree: PhD, Cell and Molecular Biology, 2012, Colorado State University

URL: http://hdl.handle.net/10217/68157

► The maintenance of blood acid-base balance is essential for survival. However, metabolic acidosis is a common clinical condition that is characterized by a significant decrease… (more)

▼ The maintenance of blood acid-base balance is essential for survival. However, metabolic acidosis is a common clinical condition that is characterized by a significant decrease in plasma pH and bicarbonate concentration. This alteration is caused by genetic or acquired defects in metabolism, in renal handling of bicarbonate, and in the excretion of titratable acid. In addition, metabolic acidosis could pose a secondary complication in patients with cachexia, trauma, uremia, end stage renal disease, osteomalacia, HIV infection and in patients with degenerative diseases. Increased renal ammoniagenesis and gluconeogenesis from plasma glutamine constitute an essential physiological response to metabolic acidosis that partially restores acid-base balance. A portion of this adaptive response is the rapid and pronounced increase in the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK) that occurs within the renal proximal convoluted tubule. Previous in vitro biochemical studies have mapped the binding of AUF1, HuR and ζ-crystallin (ζ-Cryst) to various AU-rich sequences within the 3'UTR of PEPCK mRNA. This response is reproduced in LLC-PK1-F+9C cells that are treated with acidic (pH 6.9) medium. It is mediated, in part, by stabilization of PEPCK mRNA. Here I have used a combination of approaches to characterize the dynamic interaction of trans-acting factors with the cis-acting elements in mediating the pH-responsive stabilization of PEPCK mRNA. In chapter III I show that binding of HuR and AUF1 have opposite effects on basal expression, but their co-ordinate interaction is required to mediate the pH-responsive adaptation. Consistent with this, while the individual recruitment of a chimeric protein containing the MS2 coat protein and either HuR or p40AUF1 failed to produce a pH-responsive stabilization, the concurrent expression of both chimeric proteins was sufficient to produce a pH-responsive increase in the half-life of the reporter mRNA. This study also demonstrated that HuR and AUF1 underwent profound altered post-translational modifications when LLC-PK1-F+9C cells were switched from basal to acid-pH medium conditions. In Chapter IV I went on to demonstrate that HuR makes direct interaction with PEPCK mRNA and that HuR/ AUF1 form hetero-oligomeric complex in an RNA-dependent manner. Finally in Chapter V I investigated the functional significance of the ζ-Cryst binding to the PEPCK-3'UTR. These experiments suggested that ζ-Cryst may serve as a key co-factor along with HuR and AUF1 to restrict the basal expression and that only HuR and AUF1 are required for the pH-responsive increase of PEPCK mRNA. Based upon the findings of the current study, I proposed a model depicting the co-ordinate role of HuR, AUF1 and ζ-Cryst in post-transcriptional regulation of PEPCK mRNA turnover and, more importantly in mediating the sustained pH-responsive increase of PEPCK mRNA. Under normal acid-base conditions, phosphorylated HuR, covalently modified AUF1 and ζ-crystallin are co-recruited to the 3'-UTR of PEPCK mRNA and may… Advisors/Committee Members: Curthoys, Norman P. (advisor), Nyborg, Jennifer K. (committee member), Laybourn, Paul J. (committee member), Wilusz, Carol J. (committee member).

Subjects/Keywords: 2D gel electrophoresis; RNA binding proteins; MS2 recruitment; mRNA stabilization; metabolic acidosis; siRNA knockdown

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APA (6^th Edition):

Gummadi, L. (2012). Role of HuR, AUF1 and zeta-crystallin in mediating pH-responsive increase in renal phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance in kidney cells. (Doctoral Dissertation). Colorado State University. Retrieved from http://hdl.handle.net/10217/68157

Chicago Manual of Style (16^th Edition):

Gummadi, Lakshmi. “Role of HuR, AUF1 and zeta-crystallin in mediating pH-responsive increase in renal phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance in kidney cells.” 2012. Doctoral Dissertation, Colorado State University. Accessed April 17, 2021. http://hdl.handle.net/10217/68157.

MLA Handbook (7^th Edition):

Gummadi, Lakshmi. “Role of HuR, AUF1 and zeta-crystallin in mediating pH-responsive increase in renal phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance in kidney cells.” 2012. Web. 17 Apr 2021.

Vancouver:

Gummadi L. Role of HuR, AUF1 and zeta-crystallin in mediating pH-responsive increase in renal phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance in kidney cells. [Internet] [Doctoral dissertation]. Colorado State University; 2012. [cited 2021 Apr 17]. Available from: http://hdl.handle.net/10217/68157.

Council of Science Editors:

Gummadi L. Role of HuR, AUF1 and zeta-crystallin in mediating pH-responsive increase in renal phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance in kidney cells. [Doctoral Dissertation]. Colorado State University; 2012. Available from: http://hdl.handle.net/10217/68157

Colorado State University

8. Burroughs, Chelsie Ann. Removing seminal plasma improves sex-sorting of bovine sperm.

Degree: MS(M.S.), Biomedical Sciences, 2011, Colorado State University

URL: http://hdl.handle.net/10217/49864

To view the abstract, please see the full text of the document. Advisors/Committee Members: Seidel, George E., Jr. (advisor), Graham, James K. (advisor), Curthoys, Norman P. (committee member).

Subjects/Keywords: bovine; spermatozoa; sex-sorting; seminal plasma

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APA (6^th Edition):

Burroughs, C. A. (2011). Removing seminal plasma improves sex-sorting of bovine sperm. (Masters Thesis). Colorado State University. Retrieved from http://hdl.handle.net/10217/49864

Chicago Manual of Style (16^th Edition):

Burroughs, Chelsie Ann. “Removing seminal plasma improves sex-sorting of bovine sperm.” 2011. Masters Thesis, Colorado State University. Accessed April 17, 2021. http://hdl.handle.net/10217/49864.

MLA Handbook (7^th Edition):

Burroughs, Chelsie Ann. “Removing seminal plasma improves sex-sorting of bovine sperm.” 2011. Web. 17 Apr 2021.

Vancouver:

Burroughs CA. Removing seminal plasma improves sex-sorting of bovine sperm. [Internet] [Masters thesis]. Colorado State University; 2011. [cited 2021 Apr 17]. Available from: http://hdl.handle.net/10217/49864.

Council of Science Editors:

Burroughs CA. Removing seminal plasma improves sex-sorting of bovine sperm. [Masters Thesis]. Colorado State University; 2011. Available from: http://hdl.handle.net/10217/49864

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