You searched for +publisher:"Penn State University" +contributor:("Lisa M Shantz, Committee Member").
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Penn State University
1.
Hernandez Borrero, Liz Janice.
Anti-tumor effect and destabilization of mutant p53 by CB002, a p53-pathway restoring small molecule that stimulates autophagy.
Degree: 2016, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/13038ljh216 
► Tumor suppressor p53 mediates genotoxic and cellular stress signals by controlling cell fate through transcriptional activation of genes involved in DNA repair, cell cycle arrest,…
(more)
▼ Tumor suppressor p53 mediates genotoxic and cellular stress signals by controlling cell fate through transcriptional activation of genes involved in DNA repair, cell cycle arrest, cell senescence, and apoptosis. p53 is mutated in over half of human cancers and this is associated with tumor development and chemotherapy resistance. Mutations in p53 are mostly found in the DNA-binding domain and they prevent p53 to exert its normal tumor suppressive functions. Furthermore, p53 mutations can result in gain-of-function activity, acquiring oncogenic characteristics. Therefore, altering the stability of mutant p53 protein is an attractive therapeutic strategy in cancer cells. The current project aims to identify compounds that restore the p53 pathway and modulate mutant p53 protein. Accordingly, we used a luciferase based p53 reporter to screen for small molecules that restore the p53 pathway in mutant p53-bearing cancer cells. We identified a small molecule, CB002, as a candidate for restoration of the p53 pathway in mutant p53-harboring cancer cells.
Colorectal cancer cell lines SW480 and DLD-1 were treated with different concentrations of CB002 at various time points. Cell lines exposed to CB002 showed an increase in p53 target gene expression (i.e. NOXA/DR5/P21) and apoptotic cell death markers (i.e. cleaved caspases and PARP), as early as 16 hrs. Moreover, we showed that CB002 selectively results in apoptotic cell death in cancer cells and not in WI38 normal human lung fibroblast cells as indicated by the Sub-G1 content. To explore the importance of the p53 target genes in apoptotic cell death, we knocked-down DR5 and NOXA proteins. Stable knockdown of NOXA abolished apoptotic cell death whereas DR5 did not, indicating that NOXA is required for CB002-mediated cell death. Following CB002 treatment, we observed an increase in the formation of vacuoles within treated cells. Thus, NOXA induction together with the formation of vacuoles prompted us to investigate if autophagy was playing a role in degradation of mutant p53. Autophagy induction was confirmed by LC3B conversion in cell lysates. CB002 decreased the stability of mutant p53 R175H in HCT116 and RXF393 cells. Although blocking autophagy did not rescue mutant p53 protein expression, induction of autophagy was required for cell death. We further investigated if CB002 was mediating mutant R175H p53 protein degradation through the ubiquitin proteasome system. R175H p53 mutant protein expression was largely rescued by the co-treatment of CB002 with MG132, a proteasomal inhibitor, implicating a role for the ubiquitin proteasome system. Altogether, our data suggests that CB002 stimulates apoptosis through the induction of NOXA and degrades mutant p53 R175H through the ubiquitin proteasome system. In addition, induction of autophagy by CB002 appears to be required for cell death. Hence, our results provide insight into effective p53 pathway activation through the use of small molecules.
Advisors/Committee Members: Wafik El-Deiry, Thesis Advisor/Co-Advisor, Scot R Kimball, Committee Member, Lisa M Shantz, Committee Member, Rosalyn Bryson Irby, Committee Member.
Subjects/Keywords: mutant p53; apoptosis; autophagy; p53 pathway restoration; small molecules; NOXA
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APA (6th Edition):
Hernandez Borrero, L. J. (2016). Anti-tumor effect and destabilization of mutant p53 by CB002, a p53-pathway restoring small molecule that stimulates autophagy. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/13038ljh216
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Hernandez Borrero, Liz Janice. “Anti-tumor effect and destabilization of mutant p53 by CB002, a p53-pathway restoring small molecule that stimulates autophagy.” 2016. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/13038ljh216.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Hernandez Borrero, Liz Janice. “Anti-tumor effect and destabilization of mutant p53 by CB002, a p53-pathway restoring small molecule that stimulates autophagy.” 2016. Web. 09 Mar 2021.
Vancouver:
Hernandez Borrero LJ. Anti-tumor effect and destabilization of mutant p53 by CB002, a p53-pathway restoring small molecule that stimulates autophagy. [Internet] [Thesis]. Penn State University; 2016. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/13038ljh216.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Hernandez Borrero LJ. Anti-tumor effect and destabilization of mutant p53 by CB002, a p53-pathway restoring small molecule that stimulates autophagy. [Thesis]. Penn State University; 2016. Available from: https://submit-etda.libraries.psu.edu/catalog/13038ljh216
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
2.
Jones, Victoria M.
THE ROLE OF SIRTUIN 3 IN OVARIAN CANCER METASTASIS.
Degree: 2016, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/13078vmj2
► Epithelial ovarian cancer (EOC) is highly metastatic with late-onset symptoms, making it the leading cause of death in women with gynecologic malignancies. The proposed mechanism…
(more)
▼ Epithelial ovarian cancer (EOC) is highly metastatic with late-onset symptoms, making it the leading cause of death in women with gynecologic malignancies. The proposed mechanism of metastasis involves aggressive, free-floating spheroid-like cellular aggregates that shed from the primary tumor, survive for some time in anchorage independence and eventually invade the omentum of the peritoneal cavity and nearby organs. Preliminary data revealed an upregulation of the antioxidant superoxide dismutase (SOD2) in ovarian cancer spheroids and confirmed its recently emerging, dichotomous role as a tumor promoter. We hypothesized that sirtuin 3 (SIRT3), a key regulator of SOD2, would similarly be increased in spheroids and help to promote ovarian cancer metastasis. While we observed the SIRT3 protein increase in ovarian cancer spheroids, we unexpectedly saw – upon generating SIRT3 shRNA-mediated knockdown cells – an increase in metastatic functions such as migration and proliferation. These data point to a tumor suppressive function of SIRT3 in ovarian cancer, as previously established in breast cancer. Interestingly, bioenergetics analysis characterized SIRT3 knockdown cells as having increased rates of glycolysis and glucose-dependent mitochondrial respiration. Moreover, they displayed a dependency on glucose for cell survival, while those cells with SIRT3 expression were able to survive in glucose-depleted conditions. These results may hint at clues as to why ovarian cancer spheroids increase expression of SIRT3 during the anchorage-independent stage of metastasis in the ascites fluid-containing peritoneal cavity. Efforts are underway to establish if a transient increase in SIRT3 during this anchorage-independent spheroid stage enhances cellular metabolic flexibility to rely on alternate fuel sources in situations of glucose deprivation, as may be the case in the ascites fluid.
Advisors/Committee Members: Nadine Hempel, Thesis Advisor/Co-Advisor, Kent Vrana, Committee Member, Lisa M Shantz, Committee Member, Rebecca Phaeton, Committee Member.
Subjects/Keywords: SIRT3; Sirtuin 3; Ovarian Cancer; SOD2; Spheroids; Mitochondria
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APA ·
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APA (6th Edition):
Jones, V. M. (2016). THE ROLE OF SIRTUIN 3 IN OVARIAN CANCER METASTASIS. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/13078vmj2
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Jones, Victoria M. “THE ROLE OF SIRTUIN 3 IN OVARIAN CANCER METASTASIS.” 2016. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/13078vmj2.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Jones, Victoria M. “THE ROLE OF SIRTUIN 3 IN OVARIAN CANCER METASTASIS.” 2016. Web. 09 Mar 2021.
Vancouver:
Jones VM. THE ROLE OF SIRTUIN 3 IN OVARIAN CANCER METASTASIS. [Internet] [Thesis]. Penn State University; 2016. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/13078vmj2.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Jones VM. THE ROLE OF SIRTUIN 3 IN OVARIAN CANCER METASTASIS. [Thesis]. Penn State University; 2016. Available from: https://submit-etda.libraries.psu.edu/catalog/13078vmj2
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
3.
Walsh, Erin.
Repetitive Dna Sequence Elements and Dna Polymerases Modeulate Instability in the Human Genome.
Degree: 2014, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/20639
► Abstract Repetitive DNA sequences make up over 50% of the human genome and are located in both intra- and inter-genic regions. These sequences are prone…
(more)
▼ Abstract
Repetitive DNA sequences make up over 50% of the human genome and are located in both intra- and inter-genic regions. These sequences are prone to deletion and expansion errors during replication-, repair-, and recombination-associated DNA synthesis. Additionally, alternative DNA structures that form with repetitive DNA sequences can directly impede the replication machinery during DNA synthesis. Through these mechanisms, repetitive sequences contribute to genomic instability. This thesis focuses on two types of genomic instability that occur in the human genome: microsatellite instability, which occurs primarily as a result of length alterations due to DNA strand slippage and misalignment within microsatellite repeat sequences (tandem repeats composed of one to six base pair units); and chromosomal instability, which refers to gross chromosomal aberrations, including deletions, amplifications and translocations. In particular, the goals of this thesis are to: 1) investigate the mutational behaviors of microsatellite repeats, in order to gain a better understanding of the mechanisms by which microsatellite instability is driven in the human genome; and 2) investigate the types of repetitive sequence elements and replication proteins modulating chromosomal instability at regions of the human genome known as common fragile sites (CFS).
Both DNA sequence- and cellular repair-dependent mechanisms impact the mutability of microsatellites in the human genome. As a result, the mutational behaviors of specific microsatellites located within protein or functional RNA encoding genes can directly affect gene expression levels. Additionally, cellular deficiencies of specific repair pathways can lead to increased levels of microsatellite instability genome-wide. Previous studies investigated the roles of these two mechanisms in contributing to microsatellite instability. However, a limited number of studies focused on the distinct mutational behaviors of specific microsatellites. This area of study bears biological significance towards increasing our understanding of how instability at specific repetitive sequences impacts species evolution as well as tumorigenesis.
iv
In Chapter 2, I present data from my experimental studies and computational studies performed by Dr. Guru Ananda from the laboratory of Dr. Kataryna Makova (
Penn State University,
University Park) with whom the Eckert lab collaborates. Direct comparison of computationally derived human polymorphism incidence rates and experimentally determined polymerase slippage rates within tandem repeat sequences was carried out to determine the mechanisms contributing to the mutability of mononucleotide and dinucleotide microsatellites in the human genome. Experimentally determined polymerase error frequencies (Pol EF), representing polymerase insertion and deletion errors within tandem repeat tracts, were plotted as a function of repeat length alongside a curve representing log-scale polymorphism incidence versus repeat length for mono- and dinucleotide tandem…
Advisors/Committee Members: Kristin Ann Eckert, Dissertation Advisor/Co-Advisor, Edward Joseph Gunther, Committee Member, Gregory Steven Yochum, Committee Member, Lisa M Shantz, Committee Member.
Subjects/Keywords: Specialized DNA Polymerase; Repetitive DNA Sequence; Replication Stalling; Microsatellite DNA; Common Fragile Sites
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APA ·
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APA (6th Edition):
Walsh, E. (2014). Repetitive Dna Sequence Elements and Dna Polymerases Modeulate Instability in the Human Genome. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/20639
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Walsh, Erin. “Repetitive Dna Sequence Elements and Dna Polymerases Modeulate Instability in the Human Genome.” 2014. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/20639.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Walsh, Erin. “Repetitive Dna Sequence Elements and Dna Polymerases Modeulate Instability in the Human Genome.” 2014. Web. 09 Mar 2021.
Vancouver:
Walsh E. Repetitive Dna Sequence Elements and Dna Polymerases Modeulate Instability in the Human Genome. [Internet] [Thesis]. Penn State University; 2014. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/20639.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Walsh E. Repetitive Dna Sequence Elements and Dna Polymerases Modeulate Instability in the Human Genome. [Thesis]. Penn State University; 2014. Available from: https://submit-etda.libraries.psu.edu/catalog/20639
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
4.
Kelleher, Andrew Ryan.
The regulation of mTORC1 signaling in immobilized rat hindlimb skeletal muscle.
Degree: 2014, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/22587
► Limb immobilization, limb suspension, and bed rest cause substantial loss of skeletal muscle mass, a phenomenon termed disuse atrophy. Disuse atrophy is attributed to a…
(more)
▼ Limb immobilization, limb suspension, and bed rest cause substantial loss of skeletal muscle mass, a phenomenon termed disuse atrophy. Disuse atrophy is attributed to a depression in the rates of protein synthesis in a fasted
state, and a resistance to stimulation by nutrients and other anabolic stimuli. Skeletal muscle protein synthesis is modulated by mechanistic target of rapamycin complex 1 (mTORC1) signaling, which is repressed during hindlimb immobilization. The overall goal of this research was to understand the molecular mechanisms responsible for repression of mTORC1 signaling in immobilized rat hindlimb soleus muscle. The overall hypothesis was that mTORC1 signaling is repressed in immobilized rat hindlimb skeletal muscle due to induction in the mRNA expression of two repressors of mTORC1 signaling, regulated in development and DNA damage responses (REDD) 1 and REDD2. The studies show that REDD1 and REDD2 mRNA expression are induced in association with repression of mTORC1 signaling after 1-3 days of hindlimb immobilization. Hindlimb immobilization repressed mTORC1 signaling in a fasted
state and blunted the stimulation of mTORC1 signaling in response to a bolus of leucine, a potent nutrient stimulator of mTORC1 signaling. Fixed muscle length was identified as a physiological trigger for the expression of genes associated with disuse atrophy, particularly REDD1 and REDD2. These genes were induced in soleus muscle immobilized for 3 days in a shortened position, but not in a stretched position. Aging was also associated with repression of mTORC1 signaling and induction in the mRNA expression of REDD2, while 7 days of remobilization was associated with augmented mTORC1 signaling and repression in the mRNA expression of REDD2. Collectively, these findings indicate that the mRNA expression of REDD1, and primarily REDD2 at time points longer than 3 days, is associated with changes in mTORC1 signaling under conditions of hindlimb immobilization, aging, and remobilization. REDD1 and REDD2 act as repressors and governors of the capacity for mTORC1 signaling in fasted and fed states. Furthermore, impaired PDK1 signaling to 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) and induction of REDD1 and REDD2 are associated with resistance to anabolic stimulation of mTORC1 signaling. The results support the overall hypothesis that mTORC1 signaling is repressed in immobilized rat hindlimb skeletal muscle due to induction of REDD1 and REDD2 mRNA expression.
Advisors/Committee Members: Leonard Shelton Jefferson Jr., Dissertation Advisor/Co-Advisor, Scot R Kimball, Committee Member, Christopher Martin Yengo, Committee Member, Lisa M Shantz, Committee Member, Ian Alexander Simpson, Committee Member, Donald Leon Gill, Special Member.
Subjects/Keywords: disuse atrophy; anabolic resistance; casting; REDD1; REDD2; Ddit4; Ddit4l; p70S6K1; muscle wasting; fixed muscle length
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APA ·
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MLA ·
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CSE |
Export
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Manager
APA (6th Edition):
Kelleher, A. R. (2014). The regulation of mTORC1 signaling in immobilized rat hindlimb skeletal muscle. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/22587
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Kelleher, Andrew Ryan. “The regulation of mTORC1 signaling in immobilized rat hindlimb skeletal muscle.” 2014. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/22587.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Kelleher, Andrew Ryan. “The regulation of mTORC1 signaling in immobilized rat hindlimb skeletal muscle.” 2014. Web. 09 Mar 2021.
Vancouver:
Kelleher AR. The regulation of mTORC1 signaling in immobilized rat hindlimb skeletal muscle. [Internet] [Thesis]. Penn State University; 2014. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/22587.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Kelleher AR. The regulation of mTORC1 signaling in immobilized rat hindlimb skeletal muscle. [Thesis]. Penn State University; 2014. Available from: https://submit-etda.libraries.psu.edu/catalog/22587
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
5.
Trivedi, Darshan V.
Allosteric Communication and Force Generation in Myosin Motors.
Degree: 2014, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/23282
► Muscle contraction, intracellular transport and a myriad of other mechanical functions in a cell are governed by myosin motors. Myosins utilize the chemical energy derived…
(more)
▼ Muscle contraction, intracellular transport and a myriad of other mechanical functions in a cell are governed by myosin motors. Myosins utilize the chemical energy derived from ATP hydrolysis to perform mechanical work via a cyclic interaction with actin filaments. Intricate allosteric pathways operate within the myosin molecule which leads to the coupling of different sub-domains and an efficient generation of force. The three main regions involved in this active communication are the nucleotide and actin-binding regions which are both coupled to the force-generating lever arm region. The lever arm undergoes a reversible movement defined by the recovery stroke and the powerstroke which eventually leads to the generation of force. However, the kinetic and structural details of this mechanism of force generation remain elusive. At an interface of biochemistry and biophysics, this study utilizes novel fluorophore labeling strategies combined with fluorescence spectroscopy and stopped-flow kinetics to answer these questions. Myosin V (MV) is used as a model to uncover the structural changes associated with the catalytic cycle. The rate-limiting conformational change of MV is a closed-to-open transition of the nucleotide binding region prior to the release of ADP. This study investigates the role of a specific structural element called as switch II and the magnesium (Mg) ion in the coupling between the nucleotide-and actin binding regions and their role in mediating the rate-limiting transition. Switch II was found to be critical in both these processes, while Mg played a central role in modulating the rate-limiting transition prior to ADP release. A long standing question about the precise temporal kinetics of the lever arm swing in relation to the product release steps and force generation is also unambiguously answered by this work. A novel fluorophore labeling strategy combined with stopped-flow FRET experiments unravels the kinetic mechanism of lever arm swing. The recovery stroke occurs concurrent with formation of the hydrolysis competent
state. The powerstroke occurs in two phases, a fast phase precedes phosphate release and a slow phase precedes the release of ADP. These results provide direct evidence for the order of events associated with force generation in myosins.
Advisors/Committee Members: Christopher Martin Yengo, Dissertation Advisor/Co-Advisor, Christopher Martin Yengo, Committee Chair/Co-Chair, Lisa M Shantz, Committee Member, Thomas E Spratt, Special Member, Blaise Peterson, Committee Member, William O Hancock, Committee Member.
Subjects/Keywords: Myosin; Actin; FRET; Motor Proteins
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APA (6th Edition):
Trivedi, D. V. (2014). Allosteric Communication and Force Generation in Myosin Motors. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/23282
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Trivedi, Darshan V. “Allosteric Communication and Force Generation in Myosin Motors.” 2014. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/23282.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Trivedi, Darshan V. “Allosteric Communication and Force Generation in Myosin Motors.” 2014. Web. 09 Mar 2021.
Vancouver:
Trivedi DV. Allosteric Communication and Force Generation in Myosin Motors. [Internet] [Thesis]. Penn State University; 2014. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/23282.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Trivedi DV. Allosteric Communication and Force Generation in Myosin Motors. [Thesis]. Penn State University; 2014. Available from: https://submit-etda.libraries.psu.edu/catalog/23282
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
6.
Kren, Nancy Porterfield.
Characterization of Functional Regions of OGFr.
Degree: 2015, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/26247
► The Opioid Growth Factor (OGF)-OGF receptor (OGFr) axis is present and tonically active in animal and human cancer cell lines, as well as human tumors.…
(more)
▼ The Opioid Growth Factor (OGF)-OGF receptor (OGFr) axis is present and tonically active in animal and human cancer cell lines, as well as human tumors. The OGF-OGFr pathway tonically mediates cell replication in cancer, with OGF serving as an autocrine-produced inhibitory pentapeptide. The inhibitory action of OGF on cell replication requires that OGF bind to OGFr, traffic into the nucleus to upregulate inhibitory kinases, p16 and p21 that act to stall the progression from G1-S phase of the cell cycle. Little is currently known about the functional regions required for this function.
To begin to characterize the functional regions of OGFr, mutations in OGFr identified in cancer samples were characterized. Two mutations identified in cancer samples, S378I and R444H, were characterized with respect to their effects on localization of the receptor, as well as their ability to alter DNA synthesis. R444H demonstrated a significant decrease in the nuclear/cytoplasmic ratio while S378I showed no change. Both mutations demonstrated a loss of response to OGF and the long-acting opioid receptor antagonist naltrexone (NTX) in growth assays. R444H also demonstrated a loss of inhibition in DNA synthesis, while S378I showed no significant change compared to OGFr in the BrdU assay. These data demonstrate that cancer mutations such as R444H are capable of altering the inhibitory function of OGFr, while other mutations such as S378I may be capable of altering the response to the ligand OGF or the antagonist NTX. These data contribute to the understanding of the functionally required regions of OGFr and warrant further characterization of mutations identified in cancer samples.OGFr has three nuclear localization signals (NLS) that have previously been characterized. OGFr requires at least two of the three NLSs for entry into the nucleus and for inhibition of cell proliferation. When NLSs were mutated, OGFr was excluded from the nucleus and proliferation inhibition was lost. The nuclear export of OGFr is currently unknown. In this study, endogenous OGFr, as well as exogenously expressed OGFr-EGFP, demonstrated significant nuclear accumulation in response to leptomycin B (LMB), an inhibitor of CRM1 dependent nuclear export, suggesting OGFr is exported in a CRM1 dependent manner. One leucine rich sequence fitting the consensus sequence for nuclear export signals (NES) was identified. To better characterize this sequence, the associated leucines, L217 L220 L223 and L225, were mutated to alanine in isolation as well as in combinations. All of the mutations resulted in decreased nuclear accumulation. In support of decreased nuclear localization, subNES demonstrated a loss of inhibition. When the proposed NES was fused to EGFP, NES-EGFP, it responded to LMB, indicating this sequence is capable of functioning as an export signal in isolation. To investigate why the sequence functions differently in isolation than in the context of the full-length protein, the localization of subNES was evaluated in the presence of MG132, a potent inhibitor of…
Advisors/Committee Members: Patricia McLaughlin, Dissertation Advisor/Co-Advisor, Michael Verderame, Committee Member, Ira Joseph Ropson, Committee Member, Lisa M Shantz, Committee Member, Samuel Shaomin Zhang, Committee Member.
Subjects/Keywords: OGFr; NES; Tandem Repeats
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APA ·
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MLA ·
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APA (6th Edition):
Kren, N. P. (2015). Characterization of Functional Regions of OGFr. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/26247
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Kren, Nancy Porterfield. “Characterization of Functional Regions of OGFr.” 2015. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/26247.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Kren, Nancy Porterfield. “Characterization of Functional Regions of OGFr.” 2015. Web. 09 Mar 2021.
Vancouver:
Kren NP. Characterization of Functional Regions of OGFr. [Internet] [Thesis]. Penn State University; 2015. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/26247.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Kren NP. Characterization of Functional Regions of OGFr. [Thesis]. Penn State University; 2015. Available from: https://submit-etda.libraries.psu.edu/catalog/26247
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
7.
Fino, Kristin Kelly.
Role of the Cholecystokinin-B Receptor in Proliferation of Pancreatic Cancer.
Degree: 2012, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/15980
► Pancreatic cancer ranks as the fourth most common cause of cancer-related mortality with a five-year survival rate of less than 1% and a median survival…
(more)
▼ Pancreatic cancer ranks as the fourth most common cause of cancer-related mortality with a five-year survival rate of less than 1% and a median survival of 3-6 months. Although numerous chemotherapeutic drugs have been tested in this malignancy, survival of advanced pancreatic cancer has not improved over the past several decades. Unlike other cancers, which have markers for early detection, current pancreatic cancer predictive indicators lack sensitivity and specificity. Due to the lack of effective treatment and early screening options available for this disease, identification of novel targets and approaches are desperately needed.
The human cholecystokinin-B receptor (CCK-BR) and its ligand, gastrin, are up-regulated in pancreatic cancer compared to normal tissues. Growth of pancreatic cancer is controlled by the interaction of gastrin with the CCK-BR through an autocrine and exocrine mechanism. The CCK-BR may be a promising therapeutic target. One of the goals of this study was to characterize the role of the CCK-BR in human pancreatic cancer proliferation, apoptosis, and migration. Down-regulation of the CCK-BR by RNA interference (RNAi) resulted in reduced cancer cell proliferation and an increase in apoptotic activity evidenced by increased caspase-3 activity, TUNEL positive cells, and decreased XIAP expression. In addition, cancer cell mobility was also decreased upon CCK-BR down-regulation.
An alternatively spliced isoform of the CCK-BR, the CCK-CR, retains intron 4 which is expressed as an extra 69 amino acids in the 3rd intracellular loop. Unlike the CCK-BR, this splice variant is expressed only in malignant tissues. The second goal of this study was to evaluate the CCK-CR as a diagnostic tool for detection of pancreatic cancer. Detection of CCK-CR mRNA was problematic due to low transcript abundance and the sequence similarity to the CCK-BR. CCK-CR detection proved more reliable and consistent at the protein level. The CCK-BR holds promise as a potential therapeutic target, as strategies to decrease the CCK-BR expression and activity may be beneficial for development of new treatments. Also detection of the CCK-CR could aid in development of screening options for pancreatic cancer. The data presented in this thesis give further support for both of these hypotheses.
Advisors/Committee Members: Jill P Smith, Dissertation Advisor/Co-Advisor, Dr Mark Kester, Committee Chair/Co-Chair, Rosalyn Bryson Irby, Committee Member, Lisa M Shantz, Committee Member, Hong Gang Wang, Special Member.
Subjects/Keywords: pancreatic cancer; gastrin; CCK-BR; G-protein coupled receptor; RNAi
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APA (6th Edition):
Fino, K. K. (2012). Role of the Cholecystokinin-B Receptor in Proliferation of Pancreatic Cancer. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/15980
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Fino, Kristin Kelly. “Role of the Cholecystokinin-B Receptor in Proliferation of Pancreatic Cancer.” 2012. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/15980.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Fino, Kristin Kelly. “Role of the Cholecystokinin-B Receptor in Proliferation of Pancreatic Cancer.” 2012. Web. 09 Mar 2021.
Vancouver:
Fino KK. Role of the Cholecystokinin-B Receptor in Proliferation of Pancreatic Cancer. [Internet] [Thesis]. Penn State University; 2012. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/15980.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Fino KK. Role of the Cholecystokinin-B Receptor in Proliferation of Pancreatic Cancer. [Thesis]. Penn State University; 2012. Available from: https://submit-etda.libraries.psu.edu/catalog/15980
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
8.
Raval, Manmeet Harishbhai.
MECHANISM OF CLASS III MYOSIN MEDIATED REGULATION OF ACTIN BUNDLE BASED PROTRUSIONS.
Degree: 2016, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/13601mhr15
► Class III myosins (MYO3A and MYO3B) are actin based motors which are proposed to function as transporters in parallel actin-bundle based protrusions such as stereocilia…
(more)
▼ Class III myosins (MYO3A and MYO3B) are actin based motors which are proposed to function as transporters in parallel actin-bundle based protrusions such as stereocilia of inner ear and calycal processes of photoreceptors. The first
member of the MYO3 family was identified in Drosophila photoreceptors and called NINAC (Neither inactivation nor afterpotential C) based on its role in phototransduction. It was observed that NINAC null mutant flies undergo light and age dependent retinal degeneration. The first report describing deleterious recessive mutations in MYO3A associated with inherited human progressive hearing loss (DFNB30) was published in 2002. It is believed that MYO3 dependent transport of Espin (isoforms) from the base to the tips of the stereocilia plays a crucial role in stereocilia elongation. Previous reports have hypothesized that MYO3A and MYO3B may have overlapping functions but MYO3B can only partially compensate for MYO3A. Thus, it is critically important to reveal functional differences between isoforms to determine how the loss of MYO3A leads to deafness. Using a wide-range of cell biological and biochemical approaches, this study investigated novel aspects of class III myosins and its binding partners associated with vertebrate hearing and vision. This study reports discovery of two novel MYO3 binding proteins- MORN4 and EspinL, and provide insights into the functional mechanism of each protein in association with MYO3. It was found that the tail of MYO3 has a conserved Espin1 and EspinL binding site, whereas the MORN4 binding site is distinct and present only in the MYO3A tail region. Based on structural data, a novel mechanism of motor (MYO3) mediated cargo (Espin1) activation is proposed. This work demonstrates that MYO3A is uniquely engineered to regulate formation and elongation of parallel actin bundle based protrusions which does not require rapid actin remodeling. The study also revealed a correlation between MYO3 motor activity and its ability to regulate actin protrusion formation and elongation. Interestingly, the intactness of the MYO3A extended tail region was found to be crucial for stabilizing actin protrusions. Finally, the study reports the characterization of two novel deafness causing MYO3A dominant mutations- G488E and L697W. The G488E mutation leads to an interesting phenotype of a 2-fold decrease in the maximum actin-activated ATPase rate and 2-fold increase in the actin sliding velocity. Whereas, the L697W mutation leads to a ~2-fold decreases in both the maximum actin-activated ATPase rate and actin sliding velocity. These results highlight the importance of MYO3 in vertebrate sensory structures and provides novel insights into its role in length and ultrastructure maintenance of parallel actin based protrusions.
Advisors/Committee Members: Christopher Martin Yengo, Dissertation Advisor/Co-Advisor, Christopher Martin Yengo, Committee Chair/Co-Chair, Lisa M Shantz, Committee Member, Hui-Ling Chiang, Committee Member, Colin James Barnstable, Outside Member, Scot R Kimball, Committee Member.
Subjects/Keywords: Myosins; Actin cytoskeleton; Filopodia; Stereocilia; Deafness
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Raval, M. H. (2016). MECHANISM OF CLASS III MYOSIN MEDIATED REGULATION OF ACTIN BUNDLE BASED PROTRUSIONS. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/13601mhr15
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Raval, Manmeet Harishbhai. “MECHANISM OF CLASS III MYOSIN MEDIATED REGULATION OF ACTIN BUNDLE BASED PROTRUSIONS.” 2016. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/13601mhr15.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Raval, Manmeet Harishbhai. “MECHANISM OF CLASS III MYOSIN MEDIATED REGULATION OF ACTIN BUNDLE BASED PROTRUSIONS.” 2016. Web. 09 Mar 2021.
Vancouver:
Raval MH. MECHANISM OF CLASS III MYOSIN MEDIATED REGULATION OF ACTIN BUNDLE BASED PROTRUSIONS. [Internet] [Thesis]. Penn State University; 2016. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/13601mhr15.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Raval MH. MECHANISM OF CLASS III MYOSIN MEDIATED REGULATION OF ACTIN BUNDLE BASED PROTRUSIONS. [Thesis]. Penn State University; 2016. Available from: https://submit-etda.libraries.psu.edu/catalog/13601mhr15
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
9.
Watters, Rebecca Jean.
Targeting Sphingolipid Metabolism in Natural Killer Cell Large Granular Lymphocyte Leukemia
.
Degree: 2011, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/12502
► Large granular lymphocyte (LGL) leukemia is a rare disorder characterized by clonal expansion of cytotoxic lymphocytes. LGL cells play an integral role in the immune…
(more)
▼ Large granular lymphocyte (LGL) leukemia is a rare disorder characterized by clonal expansion of cytotoxic lymphocytes. LGL cells play an integral role in the immune system and are divided into two major lineages of CD3- NK cells and CD3+ T cells that circulate throughout the blood in search of infected cells, in which they make contact through a receptor ligand to induce cell death. LGL cells are also programmed to undergo apoptosis after contact with an infected target cell; however they continue to survive in individuals with LGL leukemia. This unchecked proliferation and cytotoxicity of LGL in patients results in autoimmunity or malignancy. Although rheumatoid arthritis is the most common autoimmune condition seen in individuals with LGL leukemia, it is also associated with a wide spectrum of other autoimmune diseases. Patients may also suffer from other hematological conditions including hemolytic anemia, pure red cell aplasia, and neutropenia which lead to fatigue and recurrent bacterial infections. Currently, the only established treatment involves a low dose of an immunosuppressive regimen in combination with Methotrexate, approximately 40-50% of patients are either resistant or do not respond. As a result, no effective treatment exists, thus producing the need for novel therapeutics to be identified.
The sphingolipid metabolism network has been identified as deregulated in LGL leukemia. Lipidomic studies revealed deregulated sphingolipid metabolism as evidenced by decreased levels of overall ceramide species and increased levels of glycoslyated ceramides in leukemic NK cells, concomitant with increased glucosylceramide synthase (GCS) expression. GCS, a key enzyme of this pathway, neutralizes pro-apoptotic ceramide by transfer of a UDP-glucose. Thus, we treated both rat and human leukemic NK cells in combination with: 1) exogenous C6-ceramide incorporated into nanoliposomes in order to target mitochondria and increase physiological pro-apoptotic levels of long chain ceramide, and 2) 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of GCS. Co-administration of C6-ceramide nanoliposomes and PPMP elicited an increase in endogenous long-chain ceramide species, which led to cellular apoptosis in a synergistic manner via the mitochondrial intrinsic cell death pathway in leukemic NK cells. These results suggest that targeting ceramide metabolism may be an effective and novel strategy for treatment of NK-LGL leukemia.
Advisors/Committee Members: Thomas Loughran, Dissertation Advisor/Co-Advisor, Thomas Loughran, Committee Chair/Co-Chair, Xin Liu, Committee Chair/Co-Chair, Charles H Lang, Committee Member, Lisa M Shantz, Committee Member.
Subjects/Keywords: NK LGL Leukemia; nanoliposomes; ceramide; sphingolipid
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Watters, R. J. (2011). Targeting Sphingolipid Metabolism in Natural Killer Cell Large Granular Lymphocyte Leukemia
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/12502
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Watters, Rebecca Jean. “Targeting Sphingolipid Metabolism in Natural Killer Cell Large Granular Lymphocyte Leukemia
.” 2011. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/12502.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Watters, Rebecca Jean. “Targeting Sphingolipid Metabolism in Natural Killer Cell Large Granular Lymphocyte Leukemia
.” 2011. Web. 09 Mar 2021.
Vancouver:
Watters RJ. Targeting Sphingolipid Metabolism in Natural Killer Cell Large Granular Lymphocyte Leukemia
. [Internet] [Thesis]. Penn State University; 2011. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/12502.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Watters RJ. Targeting Sphingolipid Metabolism in Natural Killer Cell Large Granular Lymphocyte Leukemia
. [Thesis]. Penn State University; 2011. Available from: https://submit-etda.libraries.psu.edu/catalog/12502
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
10.
Kazi, Abid A.
ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
.
Degree: 2011, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/11847
► PRAS40 and DEPTOR are mTOR binding proteins that affect cell metabolism. Under catabolic conditions such as sepsis and glucocorticoid excess, there is an increase in…
(more)
▼ PRAS40 and DEPTOR are mTOR binding proteins that affect cell metabolism. Under catabolic conditions such as sepsis and glucocorticoid excess, there is an increase in total DEPTOR protein and a reduction in phosphorylation of PRAS40, suggesting that these proteins may modulate the mTOR-mediated protein synthetic response under normal and diseased conditions. The hypothesis of the present study was that knock down (KD) of PRAS40 or DEPTOR in C2C12 myocytes will increase protein synthesis via stimulating mTOR-S6K1 signaling. PRAS40 and DEPTOR KD was achieved using lentiviral particles containing shRNA to target the mouse PRAS40 and DEPTOR mRNA sequence, whereas control cells were transfected with a scrambled control shRNA. KD reduced PRAS40 and DEPTOR mRNA and protein content by 90%. PRAS40 KD did not result in increased phosphorylation of mTOR substrates or increased protein synthesis, whereas, DEPTOR KD increased both phosphorylation of mTOR kinase substrates, 4E-BP1 and S6K1, and protein synthesis. The responsiveness of PRAS40 and DEPTOR KD myocytes to anabolic (IGF-I) and catabolic (AICAR) stimuli was unaltered. Both PRAS40 and DEPTOR KD myoblasts were larger in diameter and exhibited an increased mean cell volume compared to scramble control. PRAS40 KD cells had decreased phosphorylation (S807/S811) of pRb protein. In contrast, DEPTOR KD cells had an increased phosphorylation (S807/S811) of pRb protein which is critical for the G1-S phase transition, coincident with an increased percentage of cells in the S phase. Neither PRAS40 nor DEPTOR KD altered myoblast apoptosis as evidenced by the lack of change for cleaved caspase-3. Although DEPTOR KD myoblasts did not alter autophagy as determined by a lack of change in the ratio of LC3BII/LC3BI, PRAS40 KD myoblasts had a reduced ratio of LC3BII/LC3BI. While PRAS40 KD delayed myotube formation concurrent with delayed proliferation, DEPTOR KD had the opposite effect on myogenesis and proliferation. Finally, while in vivo DEPTOR KD (~50% reduction) by electroporation into the muscle of C57/BL6 mice did not alter weight or protein synthesis in the control muscle, it prevented atrophy produced by 3 days of hindlimb immobilization, at least in part by increasing protein synthesis. Thus, our data support the hypothesis that PRAS40 and DEPTOR are important regulators of protein metabolism in myocytes and demonstrate that, while PRAS40 is required for normal myoblast growth and function, decreasing DEPTOR expression is sufficient to ameliorate the atrophic response produced by immobilization.
Advisors/Committee Members: Charles H Lang, Dissertation Advisor/Co-Advisor, Charles H Lang, Committee Chair/Co-Chair, Scot R Kimball, Committee Member, Lisa M Shantz, Committee Member, Timothy M Ritty, Committee Member.
Subjects/Keywords: protein synthesis; mTOR; knockdown; lentivirus; shRNA; sepsis; disuse atrophy
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Kazi, A. A. (2011). ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/11847
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Kazi, Abid A. “ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
.” 2011. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/11847.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Kazi, Abid A. “ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
.” 2011. Web. 09 Mar 2021.
Vancouver:
Kazi AA. ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
. [Internet] [Thesis]. Penn State University; 2011. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/11847.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Kazi AA. ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
. [Thesis]. Penn State University; 2011. Available from: https://submit-etda.libraries.psu.edu/catalog/11847
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
11.
Rennoll, Sherri Ann.
Transcriptional regulation of the c-MYC (MYC) proto-oncogene by oncogenic Wnt/β-catenin signaling in colorectal carcinomas.
Degree: 2015, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/26902
► Over 90% of sporadic colorectal cancers (CRCs) are associated with initiating mutations in components of the Wnt/β-catenin signaling pathway. These mutations result in elevated nuclear…
(more)
▼ Over 90% of sporadic colorectal cancers (CRCs) are associated with initiating mutations in components of the Wnt/β-catenin signaling pathway. These mutations result in elevated nuclear levels of the β-catenin transcriptional co-activator. In the nucleus, β-catenin associates with the T-cell factor/Lymphoid enhancer factor (TCF/Lef) family of sequence-specific transcription factors at Wnt-responsive DNA elements (WREs) to increase target gene expression. Aberrant expression of these genes by oncogenic Wnt/β-catenin signaling leads to the formation of small, benign adenomas. The c-MYC (MYC) proto-oncogene is a direct β-catenin target gene that is required for Wnt/β-catenin-mediated intestinal tumorigenesis. However, the molecular mechanisms underlying β-catenin regulation of MYC expression are not fully defined. The work described within this thesis aimed to further define these molecular mechanisms by addressing the involvement of nuclear AXIN2 and the MYC 3’ WRE in controlling oncogenic MYC gene expression.
AXIN2, a scaffolding protein and component of the Wnt/β-catenin signaling pathway, was recently demonstrated to localize to the nucleus. The function of AXIN2 within this subcellular compartment, however, was unknown. By constitutively localizing AXIN2 to the nucleus, we demonstrate that nuclear AXIN2 forms a complex with β-catenin/TCF and represses MYC gene expression by binding the MYC gene locus. These results indicate that nuclear AXIN2 functions as a molecular rheostat to maintain a “just right” level of MYC necessary for tumorigenesis in CRC. Similar to nuclear AXIN2, we find that the MYC 3’ WRE, which maps 1.4-kb downstream from the MYC transcription stop site, drives oncogenic MYC gene expression in CRC cells. Using CRISPR/Cas9 genome editing technology, we generated a knockout CRC cell line in which a single TCF binding element within the MYC 3’ WRE was deleted. Deletion of this element decreased β-catenin/TCF occupancy at the MYC 3’ WRE and MYC gene expression. The observed decrease in MYC gene expression corresponded to diminished cellular proliferation and growth of the knockout cells. Together, our studies have uncovered new mechanisms for regulation of MYC gene expression by oncogenic Wnt/β-catenin signaling, and suggest that approaches to target nuclear AXIN2 or the MYC 3’ WRE would be effective strategies for the treatment of individuals suffering from CRC.
Advisors/Committee Members: Gregory Yochum, Dissertation Advisor/Co-Advisor, Gregory Yochum, Committee Chair/Co-Chair, Laura Carrel, Committee Member, Sergei A Grigoryev, Committee Member, Lisa M Shantz, Committee Member.
Subjects/Keywords: MYC; beta-catenin; AXIN2; colorectal cancer
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Rennoll, S. A. (2015). Transcriptional regulation of the c-MYC (MYC) proto-oncogene by oncogenic Wnt/β-catenin signaling in colorectal carcinomas. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/26902
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Rennoll, Sherri Ann. “Transcriptional regulation of the c-MYC (MYC) proto-oncogene by oncogenic Wnt/β-catenin signaling in colorectal carcinomas.” 2015. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/26902.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Rennoll, Sherri Ann. “Transcriptional regulation of the c-MYC (MYC) proto-oncogene by oncogenic Wnt/β-catenin signaling in colorectal carcinomas.” 2015. Web. 09 Mar 2021.
Vancouver:
Rennoll SA. Transcriptional regulation of the c-MYC (MYC) proto-oncogene by oncogenic Wnt/β-catenin signaling in colorectal carcinomas. [Internet] [Thesis]. Penn State University; 2015. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/26902.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Rennoll SA. Transcriptional regulation of the c-MYC (MYC) proto-oncogene by oncogenic Wnt/β-catenin signaling in colorectal carcinomas. [Thesis]. Penn State University; 2015. Available from: https://submit-etda.libraries.psu.edu/catalog/26902
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
12.
Young, Megan Marie.
The Role Of Sphingosine Kinase In The Crosstalk Between Apoptosis, Autophagy And Endocytosis.
Degree: 2015, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/27408
► Autophagy is a catabolic process in which cytoplasmic components are sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes for degradation and recycling. Autophagy…
(more)
▼ Autophagy is a catabolic process in which cytoplasmic components are sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes for degradation and recycling. Autophagy functions to maintain homeostasis through the removal of cellular damage and generation of nutrients. Although autophagy and the cell death pathway of apoptosis utilize fundamentally distinct machinery to regulate cell fate, the two pathways are joined through an intricate network of molecular crosstalk. Sphingolipids represent a class of bioactive lipids that regulate various cellular processes, including apoptosis and autophagy, and thus may serve as novel regulators of the crosstalk between the two pathways. Here, we demonstrate that modulation of sphingolipid metabolism using the pan-sphingosine kinase 1/2 inhibitor, SKI-I, induces the assembly of an intracellular death-inducing signaling complex (iDISC) on autophagosomal membranes for the activation of caspase-8 and induction of apoptosis. Mechanistically, we show that the iDISC consists of two arms that recruit pro-caspase-8 to the autophagosomal membrane for oligomerization and self-activation: 1) Atg12-Atg5:FADD:caspase-8 and 2) LC3-II:p62:caspase-8. To further dissect the role of sphingosine kinase in iDISC-mediated cell death, we identified several sphingosine kinase 1 (Sphk1)-selective inhibitors to test in our system. Unexpectedly, we observed that the sphingosine-based inhibitor SK1i, but not the small molecule inhibitor PF-543, induces massive cellular vacuolization and enlargement of late endosomes and amphisomes. Notably, the loss of Sphk1 suppresses vacuolization by SK1i, and SK1i-induced endocytic vesicles fail to acquire late endosomal markers in Sphk1-deficient cells. As SK1i inhibits Sphk1 activity and the protein localizes to the enlarged vacuoles during treatment, we hypothesize that Sphk1 promotes membrane fusion independent of enzymatic activity. Furthermore, we demonstrate that the LC3 conjugation machinery and lysosome biogenesis regulate the clearance of the enlarged late endosomes. Collectively, these studies suggest that inhibition of sphingosine kinase alters autophagosomal and endosomal membrane dynamics and intracellular trafficking to regulate cell fate. Future studies will aid in identifying specific sphingolipid metabolites and/or proteins required for iDISC-dependent cell death as well as mechanistic insight into Sphk1 function in endosome trafficking. We believe that this knowledge will be critical for the implementation of autophagy-dependent apoptosis as a novel approach to enhance chemotherapeutic efficacy.
Advisors/Committee Members: Hong Gang Wang, Dissertation Advisor/Co-Advisor, Hong Gang Wang, Committee Chair/Co-Chair, Mark Kester, Committee Chair/Co-Chair, Jin Ming Yang, Committee Member, Lisa M Shantz, Committee Member, Richard Robert Young, Committee Member.
Subjects/Keywords: autophagy; apoptosis; endocytosis; iDISC; sphingosine kinase; sphingosine; sphingolipid; SK1i; SKI-I; late endosome
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Record Details
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Young, M. M. (2015). The Role Of Sphingosine Kinase In The Crosstalk Between Apoptosis, Autophagy And Endocytosis. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/27408
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Young, Megan Marie. “The Role Of Sphingosine Kinase In The Crosstalk Between Apoptosis, Autophagy And Endocytosis.” 2015. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/27408.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Young, Megan Marie. “The Role Of Sphingosine Kinase In The Crosstalk Between Apoptosis, Autophagy And Endocytosis.” 2015. Web. 09 Mar 2021.
Vancouver:
Young MM. The Role Of Sphingosine Kinase In The Crosstalk Between Apoptosis, Autophagy And Endocytosis. [Internet] [Thesis]. Penn State University; 2015. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/27408.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Young MM. The Role Of Sphingosine Kinase In The Crosstalk Between Apoptosis, Autophagy And Endocytosis. [Thesis]. Penn State University; 2015. Available from: https://submit-etda.libraries.psu.edu/catalog/27408
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
13.
Black, Adam James.
Characterization of the high-fat diet-induced effects on pre-mRNA alternative splicing in skeletal muscle: a focus on fatty acids and troponin T3.
Degree: 2018, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/14934ajb31
► Fat-enriched diets produce metabolic changes in skeletal muscle, that in turn mediate changes in gene regulation. The alternative splicing of pre-mRNA is an important part…
(more)
▼ Fat-enriched diets produce metabolic changes in skeletal muscle, that in turn mediate changes in gene regulation. The alternative splicing of pre-mRNA is an important part of gene regulation that engenders multiple protein isoforms from a single gene. Our group has previously shown that the alternative splicing of fast skeletal muscle troponin T (Tnnt3) responds to both natural and experimentally-induced changes in body mass, and that this process is impaired in obese Zucker rats, suggesting that excessive calorie consumption may impair the normal regulation of pre-mRNA alternative splicing. Therefore, the overall goal of this research was to understand if dietary factors, specifically fatty acids, affect pre-mRNA alternative splicing. The overall hypothesis was that in addition to modulating Tnnt3 pre-mRNA alternative splicing, consumption of a high-fat diet leads to transcriptome-wide changes in the pattern of alternative splicing in skeletal muscle and that these changes are mediated in part by saturated fatty acids. Studies from an exon array analysis performed on the gastrocnemius/plantaris complex of rats fed a 10% or 60% fat diet for two or eight weeks demonstrated that consumption of a high-fat diet resulted in the alternative splicing of 668 and 726 exons, respectively. Tnnt3 alternative splicing was among the top-five regulated pre-mRNAs. Further validation of these studies demonstrated that the high-fat diet-induced changes in Tnnt3 alternative splicing also occurred in rats fed a 30% fat diet across a one- to eight-week treatment period independent of changes in body mass between groups. Moreover, this effect depended on fat-type, because Tnnt3 alternative splicing occurred in response to 45% fat diets enriched with lard, but not in response to diets enriched with 45% mono- or polyunsaturated fatty acids. Collectively, these findings suggest a role for saturated fatty acids in the high-fat diet-induced modifications in Tnnt3 alternative splicing. Furthermore, addition of the saturated fatty acid palmitate to myotubes in culture induced a more robust change in Tnnt3 alternative splicing compared to the mono- or polyunsaturated fatty acids, oleate and linoleate. Treatment with a downstream metabolite of palmitate, ceramide, had effects similar to palmitate on Tnnt3 alternative splicing. Pretreatment with myriocin, an inhibitor of de novo ceramide biosynthesis, attenuated the palmitate-induced effects on alternative splicing. Furthermore, pretreatment with okadaic acid to inhibit the ceramide-induced activation of protein phosphatase 2A prevented the palmitate- and ceramide-induced Tnnt3 alternative splicing. Overall the results support the hypothesis that fat enriched diets induce changes in the pattern of alternative splicing in skeletal muscle and suggest a role for saturated fatty acids and their metabolites in mediating the changes observed in Tnnt3 alternative splicing.
Advisors/Committee Members: Scot R Kimball, Dissertation Advisor/Co-Advisor, Scot R Kimball, Committee Chair/Co-Chair, Leonard Shelton Jefferson Jr., Committee Member, Charles H Lang, Committee Member, Ralph Lauren Keil, Outside Member, Lisa M Shantz, Committee Member.
Subjects/Keywords: alternative splicing; fatty acids; Tnnt3; high-fat diet; fast skeletal muscle troponin T; skeletal muscle; ceramide
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
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to Zotero / EndNote / Reference
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APA (6th Edition):
Black, A. J. (2018). Characterization of the high-fat diet-induced effects on pre-mRNA alternative splicing in skeletal muscle: a focus on fatty acids and troponin T3. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/14934ajb31
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Black, Adam James. “Characterization of the high-fat diet-induced effects on pre-mRNA alternative splicing in skeletal muscle: a focus on fatty acids and troponin T3.” 2018. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/14934ajb31.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Black, Adam James. “Characterization of the high-fat diet-induced effects on pre-mRNA alternative splicing in skeletal muscle: a focus on fatty acids and troponin T3.” 2018. Web. 09 Mar 2021.
Vancouver:
Black AJ. Characterization of the high-fat diet-induced effects on pre-mRNA alternative splicing in skeletal muscle: a focus on fatty acids and troponin T3. [Internet] [Thesis]. Penn State University; 2018. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/14934ajb31.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Black AJ. Characterization of the high-fat diet-induced effects on pre-mRNA alternative splicing in skeletal muscle: a focus on fatty acids and troponin T3. [Thesis]. Penn State University; 2018. Available from: https://submit-etda.libraries.psu.edu/catalog/14934ajb31
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
14.
Keller, Ross Richard.
Elucidating Evolutionary Constraints of Mouse Mammary Cancer Using Adenomatous Polyposis Coli Mutations.
Degree: 2018, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/15024rrk145
► Cancer is a disease of evolution. Species evolve because mutations are passed to progeny through the germline, but somatic cells acquire mutations as well, and…
(more)
▼ Cancer is a disease of evolution. Species evolve because mutations are passed to progeny through the germline, but somatic cells acquire mutations as well, and somatic mutation can initiate cancer. In addition, established cancers themselves evolve. Within a tumor, ongoing mutagenesis creates cell-to-cell genetic diversity, allowing subpopulations to undergo natural selection in Darwinian fashion. With a surplus of cells and a staggering number of potential mutations, evolution could, in theory, empower a tumor to overcome a plethora of obstacles. But fortunately, cancer evolution is restricted. Chemical, genetic, cellular, and environmental evolutionary constraints make cancer vulnerable. To uncover novel constraints, we utilized mouse model systems of breast cancer, specifically models driven by mutations in the adenomatous polyposis coli (Apc) gene, which antagonizes oncogenic Wnt signaling.
In Chapter 2, we show that chemical and genetic constraints can direct which oncogenes are selected to cooperate in driving cancer. In mice that develop reversible, Wnt pathway-dependent mammary cancers (iWnt mice), exposure to chemical carcinogens that preferentially form adducts with either adenine (7,12 dimethylbenz(a)anthracene, DMBA) or thymine (N-ethyl-N-nitrosourea, ENU) resulted in activating mutations affecting different genes in the oncogenic Ras-Raf pathway (HrasCAA61CTA or BrafGTG636GAG, respectively). Both these mutations occur at A:T base pairs, but differ because in Hras, adenine resides on the sense strand, while in Braf, thymine does. This implicated strand-biased processes, such as strand-specific DNA repair, in mutagenesis. To confirm this, we generated DMBA- and ENU-induced mammary tumors in Apcmin mice, which inherit one nonfunctional Apc allele but routinely acquire second-hit mutations en route to mammary tumorigenesis. Exposure to each chemical led to distinct second-hit Apc mutation patterns, including strand-inverse mutation signatures at A:T sites, which precisely matched the mutation bias observed in Ras-Raf oncogenes. The findings indicate that a mutagen’s chemistry paired with DNA repair processes may have an outsized influence on which cancer driver genes are selected, independent of other pressures.
In Chapter 3, we exploited patterns of second-hit Apc driver mutations to demonstrate that ionizing radiation (IR) perturbs mutation spectra in a dose-dependent manner, implying different doses drive cancer evolution via different mechanisms. In Apcmin mice, low-dose IR exposure (1 Gy), whether administered as a one-time dose or fractionated daily doses increased mammary tumor incidence without altering the mutation spectrum compared with IR-naïve tumors. By contrast, high-dose IR (5 Gy) not only increased tumor incidence, but also shifted the mutation spectrum towards microdeletions, a putative signature of DNA double strand break (DSB) repair. Since IR is routinely used to treat breast cancer patients, we went on to determine how IR exposures impact mammary tumor cells. We introduced the Apcmin…
Advisors/Committee Members: Edward Joseph Gunther, Dissertation Advisor/Co-Advisor, Edward Joseph Gunther, Committee Chair/Co-Chair, Kristin Ann Eckert, Committee Member, Lisa M Shantz, Committee Member, James Riley Broach, Outside Member, Sarah Bronson, Committee Member.
Subjects/Keywords: Cancer; Evolution; Genetics
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Keller, R. R. (2018). Elucidating Evolutionary Constraints of Mouse Mammary Cancer Using Adenomatous Polyposis Coli Mutations. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/15024rrk145
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Keller, Ross Richard. “Elucidating Evolutionary Constraints of Mouse Mammary Cancer Using Adenomatous Polyposis Coli Mutations.” 2018. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/15024rrk145.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Keller, Ross Richard. “Elucidating Evolutionary Constraints of Mouse Mammary Cancer Using Adenomatous Polyposis Coli Mutations.” 2018. Web. 09 Mar 2021.
Vancouver:
Keller RR. Elucidating Evolutionary Constraints of Mouse Mammary Cancer Using Adenomatous Polyposis Coli Mutations. [Internet] [Thesis]. Penn State University; 2018. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/15024rrk145.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Keller RR. Elucidating Evolutionary Constraints of Mouse Mammary Cancer Using Adenomatous Polyposis Coli Mutations. [Thesis]. Penn State University; 2018. Available from: https://submit-etda.libraries.psu.edu/catalog/15024rrk145
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
15.
Mikse, Oliver.
CHARACTERIZATION OF FOXO3A AS A SUPPRESSOR OF LUNG ADENOCARCINOMA
.
Degree: 2011, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/11586
► Lung tumor development is believed to occur through a step-wise series of molecular changes that influence cell growth and survival. This process is facilitated by…
(more)
▼ Lung tumor development is believed to occur through a step-wise series of molecular changes that influence cell growth and survival. This process is facilitated by tobacco smoke which contains numerous carcinogens known to induce gene mutation and chromosomal instability (CIN). However, we have a poor understanding of the genes involved in lung cancer development. The FOXO family of transcription factors has been shown to elicit cell cycle arrest, apoptosis, and resistance to various physiologic and pathologic stresses relevant to sporadic cancer, such as DNA damage and oxidative stress. Although implicated as tumor suppressors, FOXO genetic inactivation has not been observed in human cancer. In an investigation of the two major types of non-small cell lung cancer (NSCLC) we have identified the FOXO3a gene (FOXO3a) as a novel target of deletion in human lung adenocarcinoma (LAC). Bi-allelic or homozygous deletion (HD) of FOXO3a was detected in 8 out of 33 (24.2%) mostly early-stage LAC of smokers. Another 60.6% of these tumors had losses of FOXO3a not reaching the level of HD (hereafter referred to as sub-HD). In contrast, no HD of FOXO3a was observed in 19 lung squamous cell carcinoma (LSqCC), but sub-HDs were evident. Consistent with the deletion of FOXO3a were corresponding decreases in its mRNA and protein levels in LAC. FOXO3a’s ability to induce apoptosis or cell cycle arrest in response to carcinogens was also investigated in LAC cell lines. The carcinogen (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) is strongly implicated as a cause of human lung cancer. Here we have demonstrated that FOXO3a is functionally activated and augments caspase-dependent apoptosis in cells exposed to this DNA-damaging carcinogen. Consistent with this result was the FOXO3a-dependent upregulation of pro-apoptotic effectors BIM, BNIP3 and FASL with treatment. These results implicate FOXO3a in the elimination of carcinogen-damaged cells, a role consistent with the suppression of LAC carcinogenesis, and one frequently lost through gene deletion in LAC development.
Inefficient methods of treatment lie among the main causes for poor response rates among patients who undergo lung cancer therapy. Docetaxel and vinorelbine are common anti-mitotic drugs used in second line therapy of several cancer types, including lung adenocarcinoma. These drugs include taxanes and vinca alkaloids, which induce mitotic arrest by disrupting mitotic spindle function. Previous studies in breast cancer and colon cancer cells have shown that FOXO3a stimulates apoptosis, a pro-therapeutic effect, in cells exposed to taxanes. Consequently, we investigated the role of FOXO3a in the cellular response to anti-mitotic agents in LAC cells, as this function would be lost in LAC as a consequence of gene deletion. MTS assays revealed that FOXO3a transfection causes a significant decrease in the number of viable LAC cells (both A549 and H460 cell lines) upon treatment with two different anti-mitotics, docetaxel and vinorelbine. Unlike…
Advisors/Committee Members: Chris Herzog, Dissertation Advisor/Co-Advisor, Chris Herzog, Committee Chair/Co-Chair, Tim Ritty, Committee Member, Vincent Chau, Committee Member, Leslie Joan Parent, Committee Member, Lisa M Shantz, Committee Member.
Subjects/Keywords: CDC14A; Lung Cancer; FOXO3A; Tumor Suppressor
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Mikse, O. (2011). CHARACTERIZATION OF FOXO3A AS A SUPPRESSOR OF LUNG ADENOCARCINOMA
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/11586
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Mikse, Oliver. “CHARACTERIZATION OF FOXO3A AS A SUPPRESSOR OF LUNG ADENOCARCINOMA
.” 2011. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/11586.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Mikse, Oliver. “CHARACTERIZATION OF FOXO3A AS A SUPPRESSOR OF LUNG ADENOCARCINOMA
.” 2011. Web. 09 Mar 2021.
Vancouver:
Mikse O. CHARACTERIZATION OF FOXO3A AS A SUPPRESSOR OF LUNG ADENOCARCINOMA
. [Internet] [Thesis]. Penn State University; 2011. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/11586.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Mikse O. CHARACTERIZATION OF FOXO3A AS A SUPPRESSOR OF LUNG ADENOCARCINOMA
. [Thesis]. Penn State University; 2011. Available from: https://submit-etda.libraries.psu.edu/catalog/11586
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
16.
Chin-quee, Karis P.
Breast cancer cell secretions promote a pre-metastatic niche in bone; an in vitro look
.
Degree: 2013, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/17496
► ABSTRACT Successful metastasis requires some degree of hospitality from the host organ that is metastasized. In recent years, evidence has been mounting that the primary…
(more)
▼ ABSTRACT
Successful metastasis requires some degree of hospitality from the host organ that is metastasized. In recent years, evidence has been mounting that the primary tumor contributes to adapting this remote organ to increase this hospitality. Interestingly, many in vitro experiments have shown that conditioned media from cancer cells produce physical changes which theoretically could facilitate invasion. These include perturbations in extracellular matrix and of cell to cell adhesions. The conditioned media in these in vitro experiments could be interpreted to be a proxy for either paracrine or endocrine effects, however, when these observations are coupled with evidence from in vivo experiments, it seems possible that primary tumors have remote effects on un-metastasized organs that can elicit changes in them which are pro-metastatic. We have performed a series of experiments designed to characterize and describe changes in osteoblasts (hFOB) which occur as a result of exposure to conditioned medium from breast cancer cells (MDA-MET). Functional experiments aimed at investigating whether these changes are pro-metastatic were included as a means of this characterization. Thus cell to cell adhesions, as well as the ability of the osteoblastic environment to attract MDA-MET cells were both assessed. Further, we investigated mechanistically the bases behind the observations from the functional experiments.
Methods
Confluent hFOB cells were treated with conditioned media from either MDA-MET, MDA-MB-231 HTERT-HME1 or hFOB cells 24 hrs. This treatment conditioned medium was removed, the cells washed and serum-free medium added to the hFOB cells. hFOB-conditioned medium was collected after 18hrs. This medium was then used in in vitro migration assays measuring number of MDA-MET migratory cells by labeling the cells with DNA-binding dye Cyquant; in establishing presence of collagen by western blot; in collagenase assays and in a cytokine array where antibodies to 74 cytokines were embedded on a membrane. The presence of Type 1 collagen receptors was shown by immunocytochemistry. The data were analyzed using one way ANOVA and the Student’s T test.
Results
We found that conditioned medium from hFOB cells that had been treated with MDA-MET-conditioned medium attracted more MDA-MET cells than hFOB cells pre-exposed to its own medium only. We hypothesized that Type 1 collagen fragments of specific length were the chemoattractants responsible for our observed effect. This was evidenced by an increase in collagenase in the conditioned medium of hFOB cells which had been exposed to MDA-MET-conditioned medium. The definitiveness of this evidence was aided by the inherent fidelity of the collagenase enzymes and its Type 1 collagen substrate and the distinctiveness of the product of this enzyme substrate interaction. We also showed that bacterial collagenase, which creates short collagen fragments of non-specific length removed the ability of hFOB -conditioned medium to attract MDA-MET cells. In addition, we showed that at…
Advisors/Committee Members: Henry Joseph Donahue, Dissertation Advisor/Co-Advisor, Henry Joseph Donahue, Committee Chair/Co-Chair, Andrea Manni, Committee Member, Andrea Marie Mastro, Committee Member, David Feith, Committee Member, Lisa M Shantz, Committee Member, Ralph Lauren Keil, Committee Member.
Subjects/Keywords: breast cancer metastasis; pre-metastatic niche; bone; collagen fragments; collagenase
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Chin-quee, K. P. (2013). Breast cancer cell secretions promote a pre-metastatic niche in bone; an in vitro look
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/17496
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Chin-quee, Karis P. “Breast cancer cell secretions promote a pre-metastatic niche in bone; an in vitro look
.” 2013. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/17496.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Chin-quee, Karis P. “Breast cancer cell secretions promote a pre-metastatic niche in bone; an in vitro look
.” 2013. Web. 09 Mar 2021.
Vancouver:
Chin-quee KP. Breast cancer cell secretions promote a pre-metastatic niche in bone; an in vitro look
. [Internet] [Thesis]. Penn State University; 2013. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/17496.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Chin-quee KP. Breast cancer cell secretions promote a pre-metastatic niche in bone; an in vitro look
. [Thesis]. Penn State University; 2013. Available from: https://submit-etda.libraries.psu.edu/catalog/17496
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
17.
Shi, Chenxu.
INVESTIGATIONS OF POLYAMINE FUNCTION USING TRANSGENIC MOUSE MODELS WITH SPERMIDINE SYNTHASE AND S-ADENOSYLMETHIONINE DECARBOXYLASE OVEREXPRESSION
.
Degree: 2011, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/12128
► Polyamines, including putrescine, spermidine, and spermine, are ubiquitous polycationic compounds that are essential for cell growth and differentiation. To better understand cellular function of polyamines,…
(more)
▼ Polyamines, including putrescine, spermidine, and spermine, are ubiquitous polycationic compounds that are essential for cell growth and differentiation. To better understand cellular function of polyamines, two transgenic mouse models with either widespread constitutive overexpression of spermidine synthase (SpdS) or regulated expression of S-adenosylmethionine decarboxylase (AdoMetDC) in the skin were created and characterized.
SpdS is a polyamine biosynthetic enzyme, and it transfers an aminopropyl group from decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine to produce spermidine. We generated a transgenic mouse line that overexpresses human spermidine synthase from a composite cytomegalovirus- immediate early gene enhancer/chicken β-actin promoter (CAG) in order to determine the impact of elevated SpdS activity on murine development and physiology, and to enable simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic models. CAG-SpdS mice were viable and fertile and exhibited a reduced tissue spermine:spermidine ratio that correlated with the level of SpdS activity. We also demonstrated that the combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair murine viability or lead to overt developmental abnormalities but instead normalizes the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. Finally, upon breeding of CAG-SpdS mice to MHC (α-myosin heavy chain)-AdoMetDC mice with a >100-fold increase in cardiac AdoMetDC activity, CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are unlikely to exert significant regulatory effects on cellular polyamine content and function.
Previous studies suggest that the increased levels of polyamines, especially putrescine, play a critical role in skin tumor development. AdoMetDC is a rate-limiting enzyme in the polyamine biosynthetic pathway. It catalyzes the decarboxylation of S-adenosylmethionine (AdoMet) to generate dcAdoMet that serves as the aminopropyl donor for the production of spermidine and spermine from the precursor putrescine. We utilized the Tet-off system to generate transgenic mice with regulated expression of AdoMetDC in skin basal keratinocytes directed by the Keratin 5 promoter to enhance our understanding of the role played by AdoMetDC in controlling polyamine levels and of polyamine function in epithelial carcinogenesis. We found that untreated transgenic mice displayed a 7 to 8-fold increase in epidermal and dermal AdoMetDC activity at 7 weeks of age, and a corresponding increase in the enzymatic product dcAdoMet levels. These mice also exhibited a thin fur phenotype that could be eliminated by doxycycline treatment. A skin chemical carcinogenesis experiment on these mice demonstrated that AdoMetDC overexpression resulted in a 46% reduction in tumor…
Advisors/Committee Members: David J Feith, Ph D, Dissertation Advisor/Co-Advisor, David J Feith, Committee Chair/Co-Chair, Anthony Edward Pegg, Committee Member, Lisa M Shantz, Committee Member, Edward Joseph Gunther, Committee Member, Scot R Kimball, Committee Member, Barbara Ann Miller, Committee Member.
Subjects/Keywords: Polyamines; S-adenosylmethionine decarboxylase; Spermidine synthase; Transgenic mice; Nonmelanoma skin cancer
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Shi, C. (2011). INVESTIGATIONS OF POLYAMINE FUNCTION USING TRANSGENIC MOUSE MODELS WITH SPERMIDINE SYNTHASE AND S-ADENOSYLMETHIONINE DECARBOXYLASE OVEREXPRESSION
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/12128
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Shi, Chenxu. “INVESTIGATIONS OF POLYAMINE FUNCTION USING TRANSGENIC MOUSE MODELS WITH SPERMIDINE SYNTHASE AND S-ADENOSYLMETHIONINE DECARBOXYLASE OVEREXPRESSION
.” 2011. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/12128.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Shi, Chenxu. “INVESTIGATIONS OF POLYAMINE FUNCTION USING TRANSGENIC MOUSE MODELS WITH SPERMIDINE SYNTHASE AND S-ADENOSYLMETHIONINE DECARBOXYLASE OVEREXPRESSION
.” 2011. Web. 09 Mar 2021.
Vancouver:
Shi C. INVESTIGATIONS OF POLYAMINE FUNCTION USING TRANSGENIC MOUSE MODELS WITH SPERMIDINE SYNTHASE AND S-ADENOSYLMETHIONINE DECARBOXYLASE OVEREXPRESSION
. [Internet] [Thesis]. Penn State University; 2011. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/12128.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Shi C. INVESTIGATIONS OF POLYAMINE FUNCTION USING TRANSGENIC MOUSE MODELS WITH SPERMIDINE SYNTHASE AND S-ADENOSYLMETHIONINE DECARBOXYLASE OVEREXPRESSION
. [Thesis]. Penn State University; 2011. Available from: https://submit-etda.libraries.psu.edu/catalog/12128
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
18.
Runkle, Edwin Aaron.
The Tight Junction Protein Occludin Contributes to Centrosome Separation, Cell Cycle Progression, and Barrier Properties
.
Degree: 2011, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/11595
► The transmembrane tight junction (TJ) protein occludin has been implicated in a variety of cell processes including barrier properties particularly towards small cations, regulation of…
(more)
▼ The transmembrane tight junction (TJ) protein occludin has been implicated in a variety of cell processes including barrier properties particularly towards small cations, regulation of permeability through endocytosis, and growth control. A number of studies have identified phosphorylation sites on occludin that regulate its interaction with other junctional proteins and may regulate its function. The overall goal of this dissertation was to test the hypothesis that phosphorylation of the tight junction protein occludin on Ser490 contributes to growth control and barrier function.
In this study, the Madin-Darby canine kidney (MDCK) cell line was used to study the function of a previously identified occludin phosphorylation site, Ser490. Stable transfection of the phosphomimetic Ser490 (S490D) and the non-phosphorylatable (S490A) mutants along with the wild type (WT) and empty vector (EV) were used to determine the role of occludin Ser490 in growth control. Here, we show that occludin is present at centrosomes in interphase and occludin Ser490 is phosphorylated throughout mitosis and co-localized with mitotic centrosomes in MDCK cells. Occludin S490D expressing cells showed localization of occludin at centrosomes and increased proliferation. Conversely, occludin S490A impeded centrosome separation, lowered the mitotic index following release from S-phase synchronization, and reduced proliferation. This chapter of the dissertation provides evidence that occludin is present at centrosomes and potentially regulates cell cycle progression through Ser490 phosphorylation mediated centrosome separation and subsequent mitotic entry.
In primary bovine retinal endothelial cells (BREC), phosphorylation of occludin Ser490 controls vascular endothelial growth factor (VEGF)-induced permeability. While this process is now well-defined in the retinal vasculature, an understanding of the occludin Ser490 phosphorylation contribution to small molecule permeability of epithelial cells and TJ assembly is lacking. Platelet-derived growth factor (PDGF) treatment of MDCK cells results in occludin phosphorylation and redistribution from the TJ concurrent with increases in permeability similar to the changes observed in endothelial cells treated with VEGF. Here, we verified conservation of occludin Ser490 phosphorylation in MDCK cells in response to PDGF treatment. Occludin expression was sufficient to reduce transcellular permeability to 467 Da tetramethylrhodamine (TAMRA) and arginine (Arg); however, occludin S490A further reduced TAMRA permeability while occludin S490D failed to reduce Arg permeability to the extent of wild-type (WT) and S490A. In addition, occludin S490D cells assembled the TJ in a manner similar to those expressing the empty vector, while expression of occludin WT accelerated TJ assembly. Interestingly, occludin S490A further accelerated TJ assembly. No construct affected protein content; however, in cells that stably express occludin S490A ZO-1 appeared at the TJ earlier during the recovery period. Collectively, this…
Advisors/Committee Members: David Antonetti, Dissertation Advisor/Co-Advisor, David A Antonetti, Committee Chair/Co-Chair, Leonard Shelton Jefferson Jr., Committee Chair/Co-Chair, Scot R Kimball, Committee Member, Christopher J Lynch, Committee Member, Lisa M Shantz, Committee Member, Christopher Charles Norbury, Committee Member.
Subjects/Keywords: Mitosis; Centrosomes; Tight Junctions; Occludin; Barrier Function
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APA (6th Edition):
Runkle, E. A. (2011). The Tight Junction Protein Occludin Contributes to Centrosome Separation, Cell Cycle Progression, and Barrier Properties
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/11595
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Runkle, Edwin Aaron. “The Tight Junction Protein Occludin Contributes to Centrosome Separation, Cell Cycle Progression, and Barrier Properties
.” 2011. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/11595.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Runkle, Edwin Aaron. “The Tight Junction Protein Occludin Contributes to Centrosome Separation, Cell Cycle Progression, and Barrier Properties
.” 2011. Web. 09 Mar 2021.
Vancouver:
Runkle EA. The Tight Junction Protein Occludin Contributes to Centrosome Separation, Cell Cycle Progression, and Barrier Properties
. [Internet] [Thesis]. Penn State University; 2011. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/11595.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Runkle EA. The Tight Junction Protein Occludin Contributes to Centrosome Separation, Cell Cycle Progression, and Barrier Properties
. [Thesis]. Penn State University; 2011. Available from: https://submit-etda.libraries.psu.edu/catalog/11595
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
19.
Kalapila, Aley George.
O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE - MEDIATED TOXICITY OF BIS-ELECTROPHILES: BIOCHEMICAL MECHANISMS
.
Degree: 2009, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/7914
► Bis-electrophiles are compounds that are capable of forming cross-links between two nucleophilic sites. This chemical attribute has contributed to increased genotoxicity of such compounds in…
(more)
▼ Bis-electrophiles are compounds that are capable of forming cross-links between two nucleophilic sites. This chemical attribute has contributed to increased genotoxicity of such compounds in a variety of in vitro and in vivo biological test systems. Dibromoalkanes, which are one class of bis-electrophiles, are environmental toxic agents that have been used globally as pesticides, chemical intermediates and solvents in agriculture and industry. It has been shown that the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), paradoxically augments the mutagenicity and cytotoxicity of 1,2-dibromoethane (DBE) in Escherichia coli (E.coli). DBE reacts with AGT to generate a S-(2-bromoethyl) intermediate which forms a highly reactive half-mustard at the Cys145 alkyl acceptor site. Due to its DNA-binding ability, AGT can readily facilitate the reaction between DNA and the half-mustard thus causing the formation of DNA adducts. It is likely that in E. coli these covalent AGT-DNA adducts are the major factor mediating DBE-promoted genotoxicity.
The studies described in this thesis have examined human alkyltransferase (hAGT)-mediated toxicity with other bis-electrophiles, specifically butadiene diepoxide (BDO) and epibromohydrin (EBH), which are epoxide compounds. In vitro studies were performed to investigate the reaction of hAGT with BDO and EBH. Covalent binding to DNA was observed when purified recombinant wild-type hAGT (wt-hAGT) was incubated with either BDO or EBH in the presence of an [35S]–labelled oligonucleotide. Unlike similar experiments with DBE, hAGT-DNA cross-link formation was also observed with both compounds when a mutant hAGT protein was used with its Cys145 active site residue altered to an alanine (C145A). This verified the ability of these epoxide compounds to cross-link at another residue on hAGT aside from its active site. Further analyses indicated the alternate cross-link site to be Cys150, a residue situated on the periphery of the repair protein active site pocket, in close proximity to the DNA binding motif of hAGT. Results obtained using gel electrophoresis assays led us to conclude that both BDO and EBH exhibit two different mechanisms for generating the AGT-DNA adducts. This adduct formation can occur via an initial reaction between both the protein and compound or between protein and DNA.
Studies in hAGT-expressing E.coli and CHO cells have confirmed the potentiation of toxicity by BDO, EBH and the dibromoalkanes in both model systems. With EBH and BDO, the enhancement in genotoxicity was also observed in E.coli expressing the inactive C145A-hAGT mutant, albeit not to the same degree as seen with wt-hAGT. This AGT-mediated toxicity was eliminated following expression of a C145A/C150S-hAGT double mutant indicating that covalent DNA adduct formation at the Cys150 residue were likely responsible for the increased toxicity observed in E.coli expressing the C145A mutant.
Investigation into the types of genomic alterations induced at the bacterial rpoB gene locus and the mammalian…
Advisors/Committee Members: Anthony Edward Pegg, Committee Chair/Co-Chair, Kristin Ann Eckert, Committee Member, Philip Lazarus, Committee Member, Lisa M Shantz, Committee Member, Robert G Levenson, Committee Member.
Subjects/Keywords: dibromoalkanes; dibromomethane; dibromoethane; AGT; O6-alkylguanine-DNA alkyltransferase; DNA repair; Bis-electrophiles; butadiene diepoxide; epibromohydrin; epichlorohydrin; epoxides
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APA ·
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MLA ·
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CSE |
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APA (6th Edition):
Kalapila, A. G. (2009). O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE - MEDIATED TOXICITY OF BIS-ELECTROPHILES: BIOCHEMICAL MECHANISMS
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/7914
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Kalapila, Aley George. “O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE - MEDIATED TOXICITY OF BIS-ELECTROPHILES: BIOCHEMICAL MECHANISMS
.” 2009. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/7914.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Kalapila, Aley George. “O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE - MEDIATED TOXICITY OF BIS-ELECTROPHILES: BIOCHEMICAL MECHANISMS
.” 2009. Web. 09 Mar 2021.
Vancouver:
Kalapila AG. O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE - MEDIATED TOXICITY OF BIS-ELECTROPHILES: BIOCHEMICAL MECHANISMS
. [Internet] [Thesis]. Penn State University; 2009. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/7914.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Kalapila AG. O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE - MEDIATED TOXICITY OF BIS-ELECTROPHILES: BIOCHEMICAL MECHANISMS
. [Thesis]. Penn State University; 2009. Available from: https://submit-etda.libraries.psu.edu/catalog/7914
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
20.
Balliet, Renee Marie.
CHARACTERIZATION OF UGTS ACTIVE AGAINST SUBEROYLANILIDE HYDROXAMIC ACID (SAHA): VARIATIONS IN SAHA GLUCURONIDATION ASSOCIATED WITH UGT GENETIC VARIANTS
.
Degree: 2009, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/9519
► Suberoylanilide Hydroxamic Acid (SAHA) is a histone deacetylase inhibitor used in the treatment of cutaneous T-cell lymphoma and in clinical trials for treatment of multiple…
(more)
▼ Suberoylanilide Hydroxamic Acid (SAHA) is a histone deacetylase inhibitor used in the treatment of cutaneous T-cell lymphoma and in clinical trials for treatment of multiple other cancers. Variations in patient response and toxicities have been observed; however, no underlying cause has been identified for these differences. A mechanism that could be responsible for the observed differences is altered drug metabolism. A major mode of SAHA metabolism is by glucuronidation via the UDP-glucuronosyltransferase (UGT) family of enzymes.
The UGT superfamily of enzymes catalyzes the glucuronidation of a variety of endogenous compounds such as bilirubin and steroid hormones, as well as xenobiotics such as drugs and environmental carcinogens. These enzymes are located in the endoplasmic reticulum of cells and make xenobiotics and endogenous compounds more water soluble through their conjugation to glucuronic acid in a reaction with the hydrophilic co-substrate, UDPGA. This conjugation alters the biological properties of the compound to enhance its excretion in the urine or bile and typically converts substrates into products that are less pharmacologically active. These enzymes have been associated with altered drug metabolism and correlated with patient response and toxicity.
The primary goals of this thesis include: identification of the UGTs responsible for SAHA glucuronidation, characterization of the UGTs that are most active against SAHA, in vitro studies of UGT genotype/SAHA glucuronidation phenotype association, and characterization of the promoter region of the UGT1A10 gene. To identify and characterize the UGTs active against SAHA, homogenates from UGT-overexpressing HEK293 cell lines were used. The hepatic UGTs 2B17 and 1A9 and the extra-hepatic UGTs 1A8 and 1A10 exhibited the highest overall activity against SAHA as determined by Vmax/KM (16±6.5, 7.1±2.2, 33±6.3, and 24±2.4 nL.min-1.mg UGT protein-1, respectively), with UGT2B17 exhibiting the lowest KM (300 μ
M) for SAHA of any UGT in vitro. While the UGT1A8p.Ala173Gly variant exhibited a 3-fold (P<0.005) decrease in glucuronidation activity for SAHA as compared to wild-type UGT1A8, the UGT1A8p.Cys277Tyr variant exhibited no detectable glucuronidation activity; a similar lack of detectable glucuronidation activity was observed for the UGT1A10p.Gly139Lys variant. To analyze the effects of the UGT2B17 gene deletion variant (UGT2B17*2) on SAHA glucuronidation phenotype, human liver microsomes (HLM) were analyzed for glucuronidation activity against SAHA as well as for UGT2B17 genotype. HLM from subjects homozygous for UGT2B17*2 exhibited a 45% (P<0.01) decrease in SAHA glucuronidation activity and a 75% (P<0.002) increase in KM for SAHA as compared to the HLMs from subjects homozygous for the wild-type UGT2B17*1 allele. Further stratification of the HLM glucuronidation data by gender, identified a significant difference in SAHA glucuronidation between males and females, with females having significantly less glucuronidation. Characterization of the promoter of…
Advisors/Committee Members: Philip Lazarus, Dissertation Advisor/Co-Advisor, Philip Lazarus, Committee Chair/Co-Chair, Laura Carrel, Committee Member, Lisa M Shantz, Committee Member, Christopher Herzog, Committee Member.
Subjects/Keywords: SAHA; pharmacogenetic; UGT
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Balliet, R. M. (2009). CHARACTERIZATION OF UGTS ACTIVE AGAINST SUBEROYLANILIDE HYDROXAMIC ACID (SAHA): VARIATIONS IN SAHA GLUCURONIDATION ASSOCIATED WITH UGT GENETIC VARIANTS
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/9519
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Balliet, Renee Marie. “CHARACTERIZATION OF UGTS ACTIVE AGAINST SUBEROYLANILIDE HYDROXAMIC ACID (SAHA): VARIATIONS IN SAHA GLUCURONIDATION ASSOCIATED WITH UGT GENETIC VARIANTS
.” 2009. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/9519.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Balliet, Renee Marie. “CHARACTERIZATION OF UGTS ACTIVE AGAINST SUBEROYLANILIDE HYDROXAMIC ACID (SAHA): VARIATIONS IN SAHA GLUCURONIDATION ASSOCIATED WITH UGT GENETIC VARIANTS
.” 2009. Web. 09 Mar 2021.
Vancouver:
Balliet RM. CHARACTERIZATION OF UGTS ACTIVE AGAINST SUBEROYLANILIDE HYDROXAMIC ACID (SAHA): VARIATIONS IN SAHA GLUCURONIDATION ASSOCIATED WITH UGT GENETIC VARIANTS
. [Internet] [Thesis]. Penn State University; 2009. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/9519.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Balliet RM. CHARACTERIZATION OF UGTS ACTIVE AGAINST SUBEROYLANILIDE HYDROXAMIC ACID (SAHA): VARIATIONS IN SAHA GLUCURONIDATION ASSOCIATED WITH UGT GENETIC VARIANTS
. [Thesis]. Penn State University; 2009. Available from: https://submit-etda.libraries.psu.edu/catalog/9519
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
21.
Plichta, Kristin Ann.
Mammary Epithelial Cell Subtype-Specific Analysis of Ras Pathway Activation.
Degree: 2010, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/11251
► Breast cancers can be divided into subtypes based in part on how closely the tumor cells resemble mammary epithelial cell (MEC) subtypes resident in normal…
(more)
▼ Breast cancers can be divided into subtypes based in part on how closely the tumor cells resemble mammary epithelial cell (MEC) subtypes resident in normal breast tissue. Though breast cancer subtypes differ in their aggressiveness and their response to treatment, the mechanisms by which distinct subtypes arise remain unknown. Traditionally, attempts to explain the varied clinical behavior of breast cancers rely on defining the genetic lesions harbored within individual tumors. However, recent evidence suggests that distinct breast cancer subtypes may arise from distinct MEC lineages, suggesting that some biological features of a given breast cancer subtype may be attributable to its antecedent “cell of origin”.
Elucidating whether MEC lineage impacts the biology of descendant breast cancers presents a formidable challenge. By the time a tumor is clinically detectable, breast cancers have undergone clonal evolution that precludes a reliable retrospective determination of the cell of origin. As such, prospective analyses may be required to determine whether distinct MEC subtypes yield distinct cancer subtypes. We hypothesized that distinct MEC compartments yield different phenotypes in response to an identical oncogenic stimulus. To test this possibility, we generated mouse models that permit doxycycline-dependent expression of transgenes in an MEC compartment-restricted manner and developed strategies to monitor transgene-mediated phenotypes in real-time.
As a first step toward studying malignant transformation of distinct MEC subtypes in mice, we tested experimental strategies designed to permit restriction of transgene expression to distinct MEC compartments in vitro. Transgenic mammary epithelium was partially disaggregated and propagated in 3D culture as mammary organoids, preserving the bilayered arrangement of the basal and luminal MEC compartments. Pairing a tet operator-driven H2B-eGFP reporter transgene with either a basal or luminal MEC transactivator enabled MEC compartment-restricted reporter gene expression. Notably, nuclear fluorescence resulting from H2B-eGFP expression enabled visualization of cellular dynamics within discrete MEC compartments. Through extended, multiparameter live cell imaging of organoids, time-lapse movies were generated that enabled visualization of MEC migration as well as tracking and quantitation of mitotic and apoptotic events.
Next, we adapted this system to co-express the H2B-eGFP reporter together with a tet-operator-regulated oncogenic H-RASG12V allele. Whether expressed in a basal or luminal MEC-restricted manner, H-RASG12V expression reproducibly triggered aberrant organoid growth, reflected in measureable changes in organoid size and shape. Increases in organoid size were attributable to H-RASG12V-mediated increases in cell proliferation and decreases in cell death. In addition, H-RASG12V expression in either MEC compartment drove MECs to both traverse normal compartment boundaries and adopt modes of cellular migration distinct from those encountered in the setting…
Advisors/Committee Members: Edward Joseph Gunther, Dissertation Advisor/Co-Advisor, Edward Joseph Gunther, Committee Chair/Co-Chair, Andrea Manni, Committee Member, Christopher Alan Siedlecki, Committee Member, Lisa M Shantz, Committee Member.
Subjects/Keywords: Ras; mammary organoids; breast cancer; transgenic mice; mammary epithelial cells
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Plichta, K. A. (2010). Mammary Epithelial Cell Subtype-Specific Analysis of Ras Pathway Activation. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/11251
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Plichta, Kristin Ann. “Mammary Epithelial Cell Subtype-Specific Analysis of Ras Pathway Activation.” 2010. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/11251.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Plichta, Kristin Ann. “Mammary Epithelial Cell Subtype-Specific Analysis of Ras Pathway Activation.” 2010. Web. 09 Mar 2021.
Vancouver:
Plichta KA. Mammary Epithelial Cell Subtype-Specific Analysis of Ras Pathway Activation. [Internet] [Thesis]. Penn State University; 2010. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/11251.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Plichta KA. Mammary Epithelial Cell Subtype-Specific Analysis of Ras Pathway Activation. [Thesis]. Penn State University; 2010. Available from: https://submit-etda.libraries.psu.edu/catalog/11251
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
22.
Luu, Kieu.
Positional and directional repair of O6-alkylguanine lesions by human O6-alkylguanine-DNA alkyltransferase.
Degree: 2008, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/6576
► Human O6-alkylguanine-DNA alkyltranserase (AGT) is a widely expressed DNA repair protein present in prokaryotes and eukaryotes. AGT repairs O6-alkylguanine adducts in DNA via a stoichiometric…
(more)
▼ Human O6-alkylguanine-DNA alkyltranserase (AGT) is a widely expressed DNA repair protein present in prokaryotes and eukaryotes. AGT repairs O6-alkylguanine adducts in DNA via a stoichiometric reaction that transfers the alkyl group onto an internal cysteine residue of the protein. This irreversible reaction inactivates AGT and de novo protein synthesis is required to produce more active protein. AGT protects normal cells from the mutagenic and carcinogenic effects of alkylation, but overexpression of AGT by cancer cells confers resistant to chemotherapeutic agents. The development of AGT inhibitors may prove useful in enhancing cancer chemotherapy.
O6-Benzylguanine (b6G) is a potent inactivator of AGT. We showed that a single-stranded (ss) 7-mer oligodeoxyribonucleotide (oligo) containing multiple b6G can inhibit AGT activity in vitro. The protein is more efficient at reacting with b6G residues positioned near the 5'-end compared to those positioned near the 3'-end. In vivo, oligos containing single or multiple b6G can also sensitize HT29 cells to killing by BCNU. The position of b6G in an oligo may confer some protection from serum nucleases as oligos with b6G near the 5'- or 3'-end tend to have a longer half-life.
Studies done with 16-mer oligos containing O6-methylguanine (m6G) at different positions also showed a trend in which there is an increase in the ED50 values for AGT inactivation with methylated lesions located towards the extreme 3'-end of the oligo. The ED50 values for the 16-mers with m6G positioned at the second, sixth, and eleventh nucleotide from the 5'-end were between 9-13 nM for wild type AGT. However, the ED50 values for the 16-mers with m6G positioned at the fourteenth and fifteenth nucleotide from the 5'-end were 57 nM and >50 microM, respectively. AGT repaired adducts at all positions equally well in duplex 16-mer oligos except those at positions 2 and 15. These results suggest that AGT recognizes the polarity in DNA and requires a minimum of four nucleotides 3' of an m6G lesion in order to productively bind and efficiently repair that lesion.
The preference for AGT to repair 5'-end lesions in short oligos suggested that AGT may bind DNA in an oriented manner and scans directionally (3' to 5') to identify and repair O6-alkylguanine lesions. Using a single-stranded 70-mer oligo with an m6G lesion near either end, we confirmed that AGT preferentially (3.5 fold) repaired the 5'-end m6G lesion than the 3'-end lesion. However, this preference was not apparent in double-stranded oligos.
A streptavidin/biotin-dT block was inserted between the two m6G lesions in the 70-mer oligo to impede the action of AGT. Compared to the appropriate controls, a streptavidin/biotin-dT block proximal to the 5'-end m6G in a single-stranded oligo decreased the repair of that lesion by about 2.6 fold while the repair of the 3'-end m6G was unaffected. A block distal to the 5'-end m6G decreased the repair of the 5'-end m6G to a lesser extent (1.7 fold) than the proximal block, while the repair of the 3'-end m6G…
Advisors/Committee Members: Anthony Edward Pegg, Committee Chair/Co-Chair, Kristin Ann Eckert, Committee Member, Robert G Levenson, Committee Member, Charles D Smith, Committee Member, Lisa M Shantz, Committee Member.
Subjects/Keywords: DNA repair; AGT; alkyltransferase; alkylation; chemotherapy resistance; O6-alkylguanine-DNA alkyltransferase
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Record Details
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Luu, K. (2008). Positional and directional repair of O6-alkylguanine lesions by human O6-alkylguanine-DNA alkyltransferase. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/6576
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Luu, Kieu. “Positional and directional repair of O6-alkylguanine lesions by human O6-alkylguanine-DNA alkyltransferase.” 2008. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/6576.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Luu, Kieu. “Positional and directional repair of O6-alkylguanine lesions by human O6-alkylguanine-DNA alkyltransferase.” 2008. Web. 09 Mar 2021.
Vancouver:
Luu K. Positional and directional repair of O6-alkylguanine lesions by human O6-alkylguanine-DNA alkyltransferase. [Internet] [Thesis]. Penn State University; 2008. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/6576.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Luu K. Positional and directional repair of O6-alkylguanine lesions by human O6-alkylguanine-DNA alkyltransferase. [Thesis]. Penn State University; 2008. Available from: https://submit-etda.libraries.psu.edu/catalog/6576
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
23.
Nisenberg, Oleg.
TARGETED OVEREXPRESSION OF S-ADENOSYLMETHIONINE DECARBOXYLASE IN MURINE HEARTS.
Degree: 2008, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/6593
► Myocardial disease is the most common cause of death in industrialized nations. The underlying causes of cardiac malfunction are diverse, but often relate to thromboembolic…
(more)
▼ Myocardial disease is the most common cause of death in industrialized nations. The underlying causes of cardiac malfunction are diverse, but often relate to thromboembolic events, arrhythmias, or cardiomyopathies (hypertrophic or dilated). The polyamines putrescine, spermidine and spermine are known to be essential for cell growth. Polyamines also regulate the excitability threshold and duration of action potentials in cardiomyocytes. This regulation occurs through the action at the K+ inward rectifying channels, with spermine responsible for most of the effect. Thus, studies of the interactions between polyamines and cardiac physiology should aid in the development of improved pharmaceutical agents for treating cardiac arrhythmias and hypertrophy.
The levels of decarboxylated S-adenosylmethionine (dcAdoMet), an aminopropyl donor for the synthesis of the higher polyamines spermidine and spermine, are known to be low in cardiac cells and are thought to be a limiting factor for the production of spermine. dcAdoMet is produced by S-adenosylmethionine decarboxylase (AdoMetDC), a low abundance, tightly regulated enzyme with an extremely short half-life (30 minutes to 2 hours). We have utilized the alpha-myosin heavy chain (MHC) promoter to generate transgenic mice with cardiac-specific expression of AdoMetDC to enhance our understanding of the role played by AdoMetDC in controlling polyamine levels and of the importance of polyamines in cardiac physiology.
A founder line was established with > 200-fold increase in AdoMetDC activity in the heart, resulting in decreased putrescine and spermidine levels, and increased spermine levels. Transgene expression was maximal by one week of age and remained constant into adulthood. However, the changes in polyamine levels were most pronounced during the first week of age, with a two-fold decrease in putrescine and spermidine, and a two-fold increase in spermine. Spermine returned to near control levels at three weeks of age, while the levels of putrescine and spermidine remained lower than age-matched non-transgenic littermates.
The mechanism responsible for this apparent downregulation of spermine levels in the heart was investigated by assessing expression of other metabolic enzymes in the polyamine pathway. We found that activity of ornithine decarboxylase (ODC), an enzyme that catalyzes formation of the diamine putrescine in the initial enzymatic reaction of polyamine synthesis, was decreased during the first week of age; while the activity of spermine synthase, an enzyme that catyalyzes transfer of an aminopropyl group to spermidine to form spermine, was increased. The activity of both enzymes was normalized by two weeks of age. The activity of spermidine synthase, the enzyme that catyalyzes transfer of an aminopropyl group to putrescine to form spermidine, was increased two-fold at all timepoints studied. In contrast, no detectable changes in activity, with respect to age-matched non-transgenic littermates, were found for spermine/spermidine N1-acetyl…
Advisors/Committee Members: Anthony Edward Pegg, Committee Chair/Co-Chair, Gary Alan Clawson, Committee Member, Scot R Kimball, Committee Member, Lisa M Shantz, Committee Member, Bruce A Stanley, Committee Member.
Subjects/Keywords: AdoMetDC; SAMDC; beta-adrenergic hypertrophy; isoproterenol; polyamine biosynthesis; transgenic mice
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Nisenberg, O. (2008). TARGETED OVEREXPRESSION OF S-ADENOSYLMETHIONINE DECARBOXYLASE IN MURINE HEARTS. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/6593
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Nisenberg, Oleg. “TARGETED OVEREXPRESSION OF S-ADENOSYLMETHIONINE DECARBOXYLASE IN MURINE HEARTS.” 2008. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/6593.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Nisenberg, Oleg. “TARGETED OVEREXPRESSION OF S-ADENOSYLMETHIONINE DECARBOXYLASE IN MURINE HEARTS.” 2008. Web. 09 Mar 2021.
Vancouver:
Nisenberg O. TARGETED OVEREXPRESSION OF S-ADENOSYLMETHIONINE DECARBOXYLASE IN MURINE HEARTS. [Internet] [Thesis]. Penn State University; 2008. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/6593.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Nisenberg O. TARGETED OVEREXPRESSION OF S-ADENOSYLMETHIONINE DECARBOXYLASE IN MURINE HEARTS. [Thesis]. Penn State University; 2008. Available from: https://submit-etda.libraries.psu.edu/catalog/6593
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
24.
Davidshofer, Kristine C.
ALLOSTERIC ACTIVATION OF UBIQUITIN CONJUGATING ENZYMES BY RING DOMAINS.
Degree: 2008, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/8500
► Ubiquitin is a highly conserved eukaryotic protein of 76 amino acids that is added post-translationally to other proteins or to itself by a hierarchical cascade…
(more)
▼ Ubiquitin is a highly conserved eukaryotic protein of 76 amino acids that is added post-translationally to other proteins or to itself by a hierarchical cascade of enzymes (E1, E2, and E3) in distinct pathways. Often, a polyubiquitin chain is built onto a substrate to target it for degradation by the 26S proteasome. The selection of a ubiquitin conjugating enzyme (E2) for a particular ubiquitin ligase (E3) and the subsequent transfer of ubiquitin from the E2 onto the substrate lysine are two processes that remain to be better understood. It is known that E3s harbor a limited number of conserved sequence motifs that recruit specific E2 enzymes. One such motif is known as the RING domain, comprising of amino acids that coordinate two zinc atoms in a conserved structural fold. Based on the finding that RING domains from known or putative E3 partners of Ubc7, E2-25K and Cdc34 are able to activate an intrinsic ubiquitin to ubiquitin linkage activity in these three E2 enzymes, work in this thesis uses the premise that insights on E3-E2 interactions can be obtained by studying the interactions between an E2 enzyme with isolated RING domains.
A collection of human RING domains that belong to ~100 E3s is available in our laboratory for a study toward the identification of RING domains that selectively activate Ubc7, E2-25K, or Cdc34. In work carried out in this thesis, eleven new Ubc7- and seven new E2-25K-activating RING sequences were identified. To understand the structure-function relationship of these E2-activating sequences, work in this thesis examined a RING-E2 fusion strategy to facilitate structural analysis of RING-E2 interactions. Independently of this thesis work, the gp78RING-Ubc7 fusion protein was used to obtain diffraction quality crystals from which a 2.2 Ǻ resolution structure was obtained. Using the atomic coordinates of this structure, calculation and mutational analyses were carried out to assess selected contacts at the two-protein interface. Results from this analysis identified three residues in Ubc7 (R8, L66, and V113) that contribute importantly to the binding affinity, of indicating that positive interactions make a significant contribution to E2-E3 pairings. Thus, selective E2-E3 pairings can be dictated in part by the presence of specific residues in the sequence of these proteins that supply positive interactions. Based on this analysis, it can be concluded that certain amino acids cannot readily be accommodated into the gp78-Ubc7 interface, and a negative selection process may also contribute to the stringent pairing of an E2 with an E3.
Work in this thesis also examined the catalytic function of a set of residues in Ubc7. The transfer of ubiquitin from the thiolester-linked form in an E2~Ub complex to a lysine requires the latter to be in the deprotonated form. To examine residues in Ubc7 that may assist in this deprotonation step of the reaction, work in this thesis examined the pH dependence of Ubc7 activity, with the rationale that loss of this function in a Ubc7 mutant…
Advisors/Committee Members: Vincent Chau, Committee Chair/Co-Chair, Hui Ling Chiang, Committee Member, Scot R Kimball, Committee Member, Charles H Lang, Committee Member, Lisa M Shantz, Committee Member, Mary E Truckenmiller, Committee Member.
Subjects/Keywords: RING domain; ubiquitin; ubiquitin conjugating enzyme
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APA ·
Chicago ·
MLA ·
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APA (6th Edition):
Davidshofer, K. C. (2008). ALLOSTERIC ACTIVATION OF UBIQUITIN CONJUGATING ENZYMES BY RING DOMAINS. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/8500
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Davidshofer, Kristine C. “ALLOSTERIC ACTIVATION OF UBIQUITIN CONJUGATING ENZYMES BY RING DOMAINS.” 2008. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/8500.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Davidshofer, Kristine C. “ALLOSTERIC ACTIVATION OF UBIQUITIN CONJUGATING ENZYMES BY RING DOMAINS.” 2008. Web. 09 Mar 2021.
Vancouver:
Davidshofer KC. ALLOSTERIC ACTIVATION OF UBIQUITIN CONJUGATING ENZYMES BY RING DOMAINS. [Internet] [Thesis]. Penn State University; 2008. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/8500.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Davidshofer KC. ALLOSTERIC ACTIVATION OF UBIQUITIN CONJUGATING ENZYMES BY RING DOMAINS. [Thesis]. Penn State University; 2008. Available from: https://submit-etda.libraries.psu.edu/catalog/8500
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
25.
Jacob, Kimberly Dawn.
The Role of Non-Replicative DNA Polymerases in Microsatellite Mutagenesis.
Degree: 2009, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/10186
► Microsatellites, or short tandem repeats (STR), are sequences of 1-6 base pairs per unit repeat and are found throughout the human genome. Mutations in these…
(more)
▼ Microsatellites, or short tandem repeats (STR), are sequences of 1-6 base pairs per unit repeat and are found throughout the human genome. Mutations in these sequences are sources of genetic variation and one proposed mechanism to explain these mutations is the polymerase slippage model. Although polymerase utilization of slipped DNA intermediates is a requisite step in this model, the cellular polymerase(s) responsible for microsatellite mutagenesis are unknown. In this study, we have examined the role of 2 non-replicative polymerases in microsatellite mutagenesis, Escherichia coli (E.coli) DNA Polymerase IV (Pol IV) and human DNA Polymerase β (Pol β). We investigated the loss of the E.coli dinB gene product (Pol IV) on mononucleotide and dinucleotide repeat stability. Additionally, we tried to determine the effect of knockdown of Pol β levels in human cells on mononucleotide microsatellite mutagenesis. To do this we used a Herpes Simplex Virus thymidine kinase (HSV-tk) gene episomal reporter system for microsatellite mutations. In E. coli we found that loss of dinB did not have an effect on microsatellite mutagenesis, but did cause a 4-fold reduction in the mutation rate within the coding region of the HSV-tk gene. Using an in vitro assay, we determined the mutational specificity of Pol β on mononucleotide G/C tracts. We found that Pol β makes greater than 80% of mutational events at the mononucleotide allele on both templates, which is in stark contrast to dinucleotide repeats. In human cells, we were unsuccessful at creating a knockdown of Pol β levels, however we did determine that there is an effect of lentiviral infection and selection on mutagenesis. In lentiviral infected and selected cells we observed a 3-fold reduction in overall mutation rate which was statistically significant. We also saw a shift in mutational specificity, from a bias toward coding region mutations in uninfected cells, toward mutations at the microsatellite repeat in those that were infected and selected.
Advisors/Committee Members: Kristin Ann Eckert, Dissertation Advisor/Co-Advisor, Kristin Ann Eckert, Committee Chair/Co-Chair, Sarah Bronson, Committee Member, Ralph Lauren Keil, Committee Member, Lisa M Shantz, Committee Member, Maria J Baker, Committee Member.
Subjects/Keywords: mutagenesis; poymerae beta; polymerase IV; DNA replication
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Record Details
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Jacob, K. D. (2009). The Role of Non-Replicative DNA Polymerases in Microsatellite Mutagenesis. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/10186
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Jacob, Kimberly Dawn. “The Role of Non-Replicative DNA Polymerases in Microsatellite Mutagenesis.” 2009. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/10186.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Jacob, Kimberly Dawn. “The Role of Non-Replicative DNA Polymerases in Microsatellite Mutagenesis.” 2009. Web. 09 Mar 2021.
Vancouver:
Jacob KD. The Role of Non-Replicative DNA Polymerases in Microsatellite Mutagenesis. [Internet] [Thesis]. Penn State University; 2009. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/10186.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Jacob KD. The Role of Non-Replicative DNA Polymerases in Microsatellite Mutagenesis. [Thesis]. Penn State University; 2009. Available from: https://submit-etda.libraries.psu.edu/catalog/10186
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
26.
Heakal, Yasser M.
IDENTIFICATION OF NOVEL PHARMACOLOGICAL APPROACHES TO INHIBIT
NEUROTENSIN RECEPTOR-1 MITOGENIC SIGNALING IN BREAST CANCER
CELLS
.
Degree: 2009, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/10222
► G protein coupled receptors (GPCRs) represent the largest family of cell surface receptors and serve as the primary pharmacological targets for more than thirty percent…
(more)
▼ G protein coupled receptors (GPCRs) represent the largest family of cell surface
receptors and serve as the primary pharmacological targets for more than thirty percent
of approved drugs on the market. In addition to targeting GPCRs to manage
cardiovascular and central nervous system associated disease conditions, a growing
body of literature suggests that many GPCRs are involved in cancer development,
progression and metastasis.
Neurotensin receptor 1 (NTSR1) is a GPCR that has been recently identified as a
mediator of tumorigenicity and metastasis. NTSR1, as well as its endogenous ligand,
neurotensin (NTS), are coexpressed in several types of cancer including lung,
pancreas, prostate, colon, head and neck as well as breast cancer cell lines and breast
cancer tumor samples. We have previously reported that ceramide mimetics could
inhibit breast cancer cell growth in vitro and in vivo. The direct effect of C6 ceramide on
GPCR-mediated cancer progression has not been characterized. Thus, understanding
the biochemical and biophysical regulation of NTSR1 by ceramide can help further
define NTSR1 as a novel target in breast cancer. Our results show that nanoliposomal
formulations of C6 ceramide inhibit NTSR1-mediated MDA-MB-231 breast cancer
progression (mitogenesis, migration, and matrix metalloproteinase-9 activity). In
addition, liposomal C6 ceramide inhibited NTSR1-mediated, but not phorbol 12-
myristate 13-acetate–mediated, activation of the mitogen-activated protein kinase
pathway. Mechanistically, nanoliposomal short-chain ceramide reduces NTSR1
interaction with Gáq/11 subunits within structured membrane microdomains (SMDs),
iv
consistent with diminished NTS-induced translocation of NTSR1 into membrane
microdomains.
We next hypothesized that agonist-induced receptor palmitoylation mediates
dynamic receptor translocation and signaling within SMDs. We have identified that
endogenously expressed NTSR-1 in MDA-MB-231 breast adenocarcinomas as well as
exogenously expressed NTSR-1 in HEK293T cells that do not normally express NTSR-
1 is palmitoylated at Cys 381 and Cys383. Inhibition of NTSR-1 palmitoylation in MDAMB-
231 cells as well as NTSR-1 expressing HEK293T cells diminished NTS-mediated
ERK 1/2 phosphorylation. Additionally, NTSR1 mutated at Cys381 and/or Cys383 to
serine showed diminished ERK1/2 stimulation and reduced ability to protect HEK293T
cells against apoptosis induced by serum starvation. Mechanistically, C381,383SNTSR-
1 showed reduced ability to interact with Gáq/11 and diminished localization to
SMDs, where Gáq/11 preferentially resides.
Based on these observations, we next hypothesized that ceramide effects on NTSR-
1 localization and signaling could be in part mediated through interference with NTSR-1
palmitoylation/depalmitoylation cycle. Our results suggest that ceramide could either
inhibit NTSR-1 palmitoylation through direct biochemical or biophysical mechanism(s) or
through acceleration of NTSR-1 deplamitoylation.
Collectively, our findings suggest that liposomal short-chain C6 ceramide can…
Advisors/Committee Members: Dr Mark Kester, Dissertation Advisor/Co-Advisor, Mark Kester, Committee Chair/Co-Chair, Lisa M Shantz, Committee Member, Jong Kak Yun, Committee Member, Chris Herzog, Committee Member, Melvin Lee Billingsley, Committee Member.
Subjects/Keywords: Breast Cancer; Neurotensin; Ceramide; GPCR
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Heakal, Y. M. (2009). IDENTIFICATION OF NOVEL PHARMACOLOGICAL APPROACHES TO INHIBIT
NEUROTENSIN RECEPTOR-1 MITOGENIC SIGNALING IN BREAST CANCER
CELLS
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/10222
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Heakal, Yasser M. “IDENTIFICATION OF NOVEL PHARMACOLOGICAL APPROACHES TO INHIBIT
NEUROTENSIN RECEPTOR-1 MITOGENIC SIGNALING IN BREAST CANCER
CELLS
.” 2009. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/10222.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Heakal, Yasser M. “IDENTIFICATION OF NOVEL PHARMACOLOGICAL APPROACHES TO INHIBIT
NEUROTENSIN RECEPTOR-1 MITOGENIC SIGNALING IN BREAST CANCER
CELLS
.” 2009. Web. 09 Mar 2021.
Vancouver:
Heakal YM. IDENTIFICATION OF NOVEL PHARMACOLOGICAL APPROACHES TO INHIBIT
NEUROTENSIN RECEPTOR-1 MITOGENIC SIGNALING IN BREAST CANCER
CELLS
. [Internet] [Thesis]. Penn State University; 2009. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/10222.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Heakal YM. IDENTIFICATION OF NOVEL PHARMACOLOGICAL APPROACHES TO INHIBIT
NEUROTENSIN RECEPTOR-1 MITOGENIC SIGNALING IN BREAST CANCER
CELLS
. [Thesis]. Penn State University; 2009. Available from: https://submit-etda.libraries.psu.edu/catalog/10222
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
27.
Shah, Sandeep.
Microsatellites as inducers of genome instability: Studies into the mechanisms of replication and repair of repetitive DNA
.
Degree: 2010, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/10468
► Genomic instability is a hallmark of tumor initiation and progression. A tumor genome can contain both gross chromosomal alterations as well as multiple submicroscopic nucleotide…
(more)
▼ Genomic instability is a hallmark of tumor initiation and progression. A tumor genome can contain both gross chromosomal alterations as well as multiple submicroscopic nucleotide changes. Indeed, it is well established that multiple mutations are necessary for a cell to become tumorigenic. In this dissertation, I have studied two major cellular processes that, under normal circumstances, largely suppress the initial events that favor these alterations. These two processes are DNA replication, which duplicates the genome in order to fully distribute all genetic information to two daughter cells, and DNA mismatch repair, which corrects a subset of errors that occur during replication.
DNA replication is an extremely accurate process where, on average, mutations occur once every 109-1010 base pairs during cell division. The central proteins for replication are DNA polymerases which travel along the template DNA while extending nascent DNA by nucleotide addition. While the vast majority of DNA is in the right-handed double helix form, many other conformations can exist within particular sequence arrangements. These non-canonical structures can impede the polymerase and stall replication until secondary mechanisms restart the process. Specific chromosomal areas have been found that are especially prone to replicative stress. These areas, termed common fragile sites (CFS), exhibit chromosomal gaps and breaks during replicative challenge and may represent a mechanism for initiating genomic instability. Chromosomal breaks are a prerequisite for alterations such as translocations, large deletions, and amplifications found in advanced tumor cells. CFS are also among the last regions to be replicated during culture of healthy untreated cells. Studies have determined that the majority of CFS are AT rich, a property believed to cause increased torsional stress and DNA secondary structure formation. This finding has lead to the hypothesis that CFS expression (breakage) may be due, in part, to stalling of the polymerase complex.
Although DNA replication is a very accurate process on average, certain regions – e.g., microsatellites – have a very high error rate. Microsatellites are short, repetitive elements in the DNA consisting of 1-6 bases per repeat unit. The repetitive nature of these sequences can provoke length alterations due to strand slippage events during replication. The human DNA mismatch repair (MMR) system, a specialized repair mechanism, plays a major role in post-replicative repair of DNA polymerization errors. Loss of this pathway results in hereditary cancers characterized by microsatellite instability.
In this dissertation, I first describe our studies of hPMS2, a component of MMR, and observe its role in error correction of mono-, di-, and tetranucleotide repeat motifs. Alterations at mono- and dinucleotide microsatellites have been extensively used to diagnose hereditary nonpolyposis colorectal cancer, while tetranucleotide microsatellites have been utilized for the detection of…
Advisors/Committee Members: Kristin Ann Eckert, Dissertation Advisor/Co-Advisor, Kristin Ann Eckert, Committee Chair/Co-Chair, Gavin Peter Robertson, Committee Member, Sergei A Grigoryev, Committee Member, Leslie Joan Parent, Committee Member, Lisa M Shantz, Committee Member.
Subjects/Keywords: primer extension; polymerase; WRN; replication; FRA16D; PMS2; Mismatch Repair; Fragile sites; HNPCC; mutation bias
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Shah, S. (2010). Microsatellites as inducers of genome instability: Studies into the mechanisms of replication and repair of repetitive DNA
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/10468
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Shah, Sandeep. “Microsatellites as inducers of genome instability: Studies into the mechanisms of replication and repair of repetitive DNA
.” 2010. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/10468.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Shah, Sandeep. “Microsatellites as inducers of genome instability: Studies into the mechanisms of replication and repair of repetitive DNA
.” 2010. Web. 09 Mar 2021.
Vancouver:
Shah S. Microsatellites as inducers of genome instability: Studies into the mechanisms of replication and repair of repetitive DNA
. [Internet] [Thesis]. Penn State University; 2010. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/10468.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Shah S. Microsatellites as inducers of genome instability: Studies into the mechanisms of replication and repair of repetitive DNA
. [Thesis]. Penn State University; 2010. Available from: https://submit-etda.libraries.psu.edu/catalog/10468
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
28.
Ackermann, Joseph Michael.
NOVEL APPROACHES TO MODULATE ORNITHINE DECARBOXYLASE ACTIVITY AND TO DETERMINE A ROLE FOR ORNITHINE DECARBOXYLASE IN CELL TRANSFORMATION.
Degree: 2008, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/6533
► Ornithine decarboxylase (ODC) is known to have an important role in cell transformation, but that role is not well understood. The scientific and clinical value…
(more)
▼ Ornithine decarboxylase (ODC) is known to have an important role in cell transformation, but that role is not well understood. The scientific and clinical value of reducing the activity of ODC, a key enzyme in the biosynthesis of polyamines, is well appreciated. Polyamines are necessary components for cell growth and manipulation of polyamine homeostasis may be an effective strategy for the treatment of a number of disorders, including neoplastic diseases. Indeed, -difluoromethylornithine (DFMO), a specific enzyme-activated irreversible inhibitor of ODC is currently in clinical trials as a potential chemopreventative/therapeutic agent and is clinically used for other disorders. However, the effectiveness of DFMO is limited by the drug's pharmacokinetic profile and the unique regulation of ODC.
Targeting ODC mRNA may be an effective method to reduce ODC activity due to the enzyme's extensive regulation at the translational and protein levels. The half-life of ODC is among the shortest of any known protein. Due to the rapid turnover of ODC protein, cells are reliant on translation of new protein to maintain or elevate ODC activity. A number of reports have indicated that little to no change in ODC mRNA content is often detected upon substantial increases or decreases in ODC protein content and activity. Reversible inhibitors of ODC protein have a disadvantage because they can stabilize ODC from degradation. The initial inhibition of ODC leads to increased ODC transcription and active ODC can accumulate in the cells. Conversely, the efficacy of irreversible inhibitors may be limited by the short half-life of ODC. Therefore targeting ODC mRNA, rather than protein, may be a better strategy.
An approach to develop an effective 10-23 DNAzyme and hammerhead ribozyme against ODC is described in these studies. DNAzymes and ribozymes bind to a cognate RNA substrate via Watson-Crick base pairing hybridization arms and subsequently catalyze cleavage of a phosphodiester bond of the RNA. The major difference between the two is the base composition; DNAzymes are composed of DNA, ribozymes of RNA. DNAzymes capable of cleaving the target ODC RNA were identified in vitro and further characterized by the effect each had on ODC protein and activity levels using in vitro translated ODC RNA. ODC protein levels and activity correlated well with the RNA cleavage activity of the DNAzyme. A DNAzymes that exhibited good activity was optimized for use in cell culture studies. The length of the DNAzyme hybridization arms was altered and kinetics were performed to identify the most catalytically efficient configuration. One DNAzyme, DZ IV, with equal length arms of nine nucleotides proved to be the most catalytically efficient. In HEK 293 cells, DZ IV was able to reduce the amount of translated ODC protein resulting in ~80% reduction in ODC activity—a statistically significant enhancement over the antisense effect of a catalytically inactive DNAzyme.
The effectiveness of DZ IV was evaluated an in vivo model with clinical…
Advisors/Committee Members: Anthony Edward Pegg, Committee Chair/Co-Chair, Melvin Lee Billingsley, Committee Member, Mark Kester, Committee Member, Lisa M Shantz, Committee Member, Charles D Smith, Committee Member, Kent Eugene Vrana, Committee Member.
Subjects/Keywords: polyamines; ornithine decarboxylase; gene silencing; DNazyme; ribozyme; antisense oligodeoxynucleotides; difluoromethylornithine; DFMO; resonance energy transfer; RET; FRET; catalytic nuleic acids
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Record Details
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Ackermann, J. M. (2008). NOVEL APPROACHES TO MODULATE ORNITHINE DECARBOXYLASE ACTIVITY AND TO DETERMINE A ROLE FOR ORNITHINE DECARBOXYLASE IN CELL TRANSFORMATION. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/6533
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Ackermann, Joseph Michael. “NOVEL APPROACHES TO MODULATE ORNITHINE DECARBOXYLASE ACTIVITY AND TO DETERMINE A ROLE FOR ORNITHINE DECARBOXYLASE IN CELL TRANSFORMATION.” 2008. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/6533.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Ackermann, Joseph Michael. “NOVEL APPROACHES TO MODULATE ORNITHINE DECARBOXYLASE ACTIVITY AND TO DETERMINE A ROLE FOR ORNITHINE DECARBOXYLASE IN CELL TRANSFORMATION.” 2008. Web. 09 Mar 2021.
Vancouver:
Ackermann JM. NOVEL APPROACHES TO MODULATE ORNITHINE DECARBOXYLASE ACTIVITY AND TO DETERMINE A ROLE FOR ORNITHINE DECARBOXYLASE IN CELL TRANSFORMATION. [Internet] [Thesis]. Penn State University; 2008. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/6533.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Ackermann JM. NOVEL APPROACHES TO MODULATE ORNITHINE DECARBOXYLASE ACTIVITY AND TO DETERMINE A ROLE FOR ORNITHINE DECARBOXYLASE IN CELL TRANSFORMATION. [Thesis]. Penn State University; 2008. Available from: https://submit-etda.libraries.psu.edu/catalog/6533
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Penn State University
29.
Tuckow, Alexander Paul.
CONTROL OF SKELETAL MUSCLE PROTEIN SYNTHESIS:
FUNCTION AND REGULATION OF EUKARYOTIC INITIATION FACTOR 2B EPSILON
.
Degree: 2010, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/11619
► Eukaryotic initiation factor 2B (eIF2B) is a heteropentameric complex that functions as a guanine nucleotide exchange factor (GEF) toward its substrate, eIF2. The activity of…
(more)
▼ Eukaryotic initiation factor 2B (eIF2B) is a heteropentameric complex that functions as a guanine nucleotide exchange factor (GEF) toward its substrate, eIF2. The activity of eIF2B is tightly regulated and is rate controlling with regard to global protein synthesis by impacting the rate of the initiation phase of mRNA translation. Alterations in skeletal muscle eIF2B activity and expression of its catalytic epsilon subunit (eIF2Bε) have been observed in a number of physiological conditions where protein synthesis is altered. One objective of the dissertation was to directly examine the effects of exogenously expressed eIF2Bε on GEF activity and protein synthetic rates in rat skeletal muscle in vivo. Using electro gene transfer, a plasmid encoding FLAG-eIF2Bε was transfected into the tibialis anterior (TA) of one hindlimb while the contralateral TA received a control plasmid. Overexpression of the eIF2Bε subunit resulted in increased GEF activity in TA homogenates of healthy rats demonstrating that the expressed protein can enhance catalytic activity. In an effort to restore a deficit in eIF2B activity, an established model of chronic sepsis was utilized in which skeletal muscle eIF2B activity is known to be impaired. Ectopic expression of FLAG-eIF2Bε in the TA rescued the sepsis-induced deficit in eIF2B GEF activity and muscle protein synthesis. The results demonstrate that modulation of the expression of eIF2Bε may be sufficient to correct deficits in skeletal muscle protein synthesis associated with sepsis and possibly other muscle wasting conditions.
Unexpectedly, attempts at longer-term experiments revealed difficulty in maintaining elevated expression of eIF2Bε in rat skeletal muscle beyond a few days despite the ability to express other genes in muscle for several weeks. Thus, a second objective of the research was to identify the mechanism(s) accounting for the rapid loss of exogenously expressed eIF2Bε protein. Evidence obtained in this regard points to a covalent modification of the eIF2Bε protein (ubiquitination) that targets it for proteasome-mediated degradation. Following co-immunoprecipitation of eIF2Bε with ubiquitin from lysates of C2C12 myoblasts treated with proteasome inhibitor, tandem mass spectrometry analysis was performed to locate posttranslational modifications on the eIF2Bε protein. Although the site(s) of ubiquitin modification have not yet been identified, the mass spectrometry analysis resulted in the identification of two novel phosphorylation sites in the rat eIF2Bε protein. The newly identified mechanism for regulating eIF2Bε expression via the ubiquitin-proteasome system may be particularly important with regard to its potential role in muscle wasting conditions where protein synthesis is impaired.
Statins are a widely prescribed and effective class of lipid lowering drugs with many beneficial effects; however, one of the less desirable side effects of statin use is the incidence of…
Advisors/Committee Members: Leonard Shelton Jefferson Jr., Dissertation Advisor/Co-Advisor, Leonard Shelton Jefferson Jr., Committee Chair/Co-Chair, Scot R Kimball, Committee Member, Charles H Lang, Committee Member, Lisa M Shantz, Committee Member, Willard M Freeman, Committee Member.
Subjects/Keywords: translation initiation; protein synthesis; skeletal muscle
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APA (6th Edition):
Tuckow, A. P. (2010). CONTROL OF SKELETAL MUSCLE PROTEIN SYNTHESIS:
FUNCTION AND REGULATION OF EUKARYOTIC INITIATION FACTOR 2B EPSILON
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/11619
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Tuckow, Alexander Paul. “CONTROL OF SKELETAL MUSCLE PROTEIN SYNTHESIS:
FUNCTION AND REGULATION OF EUKARYOTIC INITIATION FACTOR 2B EPSILON
.” 2010. Thesis, Penn State University. Accessed March 09, 2021.
https://submit-etda.libraries.psu.edu/catalog/11619.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Tuckow, Alexander Paul. “CONTROL OF SKELETAL MUSCLE PROTEIN SYNTHESIS:
FUNCTION AND REGULATION OF EUKARYOTIC INITIATION FACTOR 2B EPSILON
.” 2010. Web. 09 Mar 2021.
Vancouver:
Tuckow AP. CONTROL OF SKELETAL MUSCLE PROTEIN SYNTHESIS:
FUNCTION AND REGULATION OF EUKARYOTIC INITIATION FACTOR 2B EPSILON
. [Internet] [Thesis]. Penn State University; 2010. [cited 2021 Mar 09].
Available from: https://submit-etda.libraries.psu.edu/catalog/11619.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Tuckow AP. CONTROL OF SKELETAL MUSCLE PROTEIN SYNTHESIS:
FUNCTION AND REGULATION OF EUKARYOTIC INITIATION FACTOR 2B EPSILON
. [Thesis]. Penn State University; 2010. Available from: https://submit-etda.libraries.psu.edu/catalog/11619
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
. 
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