Theses and Dissertations

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Open Access
Protein expression of HER-2, CAV-1 and ER alpha in canine mammary tumors and canine osteosarcoma
(Colorado State University. Libraries, 2007) Flint, Alfred Frederick, author; Hanneman, William H., advisor
Human Epidermal Growth Factor 2 (HER-2, neu or erbB-2) is a protein that influences cell proliferation, morphological differentiation, and cell motility. Additionally, over expression of HER-2 has been shown to promote growth and invasion of cells of mammary neoplasia in vivo. Over expression of HER-2 has been identified in 25-30% of human and canine mammary neoplasms and osterosarcomas though the prognostic significance remains unclear. HER-2 over expression in human breast cancer correlates with a more aggressive tumor type, poor prognosis and resistance to chemotherapeutic agents. However, patients with breast cancer over-expressing HER-2 have benefited from anti-HER-2 therapy. By targeting HER-2, cell proliferation is subsequently inhibited by blocking intracellular signaling with direct targets to the cell cycle machinery. The studies presented examine the complex protein interactions of HER-2 in mammary neoplasia and osteosarcoma in canine patients. Real-time RT-PCR was used to evaluate HER-2 expression in 7 canine OSA cell lines and 10 canine OSA tissue samples. HER-2 is significantly over expressed in 86% (6/7) of the cell lines and 40% (4/10) of the OSA tissues samples. Given the importance of HER-2 in human breast cancer, the finding of HER-2 over expression in canine OSA may be important in further understanding the pathogenesis and possible therapies of OSA. Histomorphologic characterization and immunohistochemical analysis of HER-2, caveolin 1 (CAV-1), and estrogen receptor alpha (ERα) was performed on 144 canine mammary tumors from 44 different breeds. HER-2 was over expressed (score 3) in 23.4% (85/137) of the lesions. Patients with lesions over-expressing HER-2 had a 109d decrease in the median time to reoccurrence and a 276d decrease in median survival time. CAV-1 showed little or no expression in 31% (45/139) of lesions. However when CAV-1 was over expressed (score 2-3) patients had a decrease in the median time to reoccurrence of 236d and a decrease in median survival time of 292d. Finally, ERα expression in the cytoplasm was correlated to lesions that were classified as benign. The immunohistochemical evaluation of HER-2, CAV-1, and ERα support their use in prognostic evaluation.
Open Access
Primary and secondary metabolism in Centaurea maculosa and their potential roles in invasion biology
(Colorado State University. Libraries, 2008) Broeckling, Corey D., author; Vivanco, Jorge M., advisor
Centaurea maculosa is plant species native to Eurasia which has become invasive in North America, in part through allelopathic behavior. Allelopathy remains a highly debated subject, and a more firm understanding is necessary. C. maculosa is reported to secrete catechin as an allelochemical that is toxic to North American native plants. In this dissertation, a novel colorimetric assay for use in detection of catechin from soils is described and validated. This assay is highly sensitive, selective, and fast, which should allow for more detailed measures of catechin under field conditions, and ultimately a better understanding of the variation in catechin accumulation. To increase the depth of understanding of catechin biosynthesis, I report the cloning and characterization of C. maculosa dihydroflavonol reductase (CmDFR), a gene very likely to be necessary for catechin biosynthesis. To expand our view of metabolism beyond catechin, metabolome analysis is applied to field collected plant material, and demonstrate that the physiology of C. maculosa varies with the surrounding plant community -- plants growing in patches with high C. maculosa density tend to be accumulate higher levels of secondary metabolites than plants growing in the company of few conspecifics. Finally, the results of a study that clarifies the role of root exudates in structuring the soil fungal community are presented in the context of invasion biology.
Open Access
In-vivo investigation of resveratrol as a preventive for radiation-induced acute myeloid leukemia
(Colorado State University. Libraries, 2008) Carsten, Ronald E., author; Ullrich, Robert L., advisor
Resveratrol has been shown to have cancer preventive properties. It modulates a wide range of molecular targets including those involved with induction of apoptosis and cell cycle arrest in a concentration dependent fashion. This study was designed to investigate resveratrol's ability to reduce radiation-induced chromosome aberrations and PU.1 gene loss in mouse bone marrow cells. Loss of 1 PU.1 gene and missense mutation of the remaining allele leads to acute myeloid leukemia in CBA mice.
Open Access
Role of gamma-aminobutyric acid (GABA) in hypothalamic nuclear development
(Colorado State University. Libraries, 2008) McClellan, Kristy Michelle, author; Tobet, Stuart A., advisor
The hypothalamus is involved in energy balance, appetite regulation, stress and anxiety, reproduction including sex behavior, and aggression. It is divided into cell groups based on cell staining, functional similarities and projections. This dissertation looks at the development of three of these cell groups, the ventromedial nucleus (VMN), the paraventricular nucleus (PVN), and the arcuate nucleus (ARC). The VMN and PVN have a unique pattern of GABA expression in elements surrounding the nuclei, with a void of GABA within the region of the developing nuclei. The ARC, on the other hand, has dense GABAergic immunoreactive elements throughout the nucleus. Because of the expression pattern of GABA immunoreactivity surrounding the VMN and PVN, it is likely that GABA is acting as a boundary cue to influence migrating neurons. Chapter 2 reviews the development of the VMN, including a discussion of the heterogeneity of the nucleus, a description of what cues are involved in cell migration, and descriptive information on the directions of cell movement during development. Chapter 3 examines the role of GABA on cell migration within the VMN and ARC. A live slice culture system allowed visual tracking of cell movement in the VMN and ARC. There was a difference between the average movement speeds of cells in the VMN vs. cells in the ARC, and upon addition of GABA receptor antagonists to the slices, VMN cells increased in speed while ARC cells did not change. In mice lacking functional GABAB receptors there was a change in cell positions of neurons containing immunoreactive estrogen receptor (ER)α, which may be due to changes in cell movements and migration. There was no change in amount of cell positions of immunoreactive ERα cells in the ARC. Chapter 4 looks at the role of GABA and brain derived neurotrophic factor (BDNF) in PVN development. There was a sex-selective effect in GABAB R1 subunit knockout mice of positions of cells containing immunoreactive ERÎα, indicating that females may be particularly more susceptible to changes in GABA signaling as it may be influencing the final positions of cells. There was also a decrease in BDNF expression in GABABR1 subunit knockout mice, suggesting that GABA may play a role in cell differentiation. In conclusion, several lines of evidence indicate roles for GABA in the development of the hypothalamus, in particular, within the VMN and PVN.
Open Access
Serial protein misfolding cyclic amplification (sPMCA) to detect surrogate markers for chronic wasting disease in surface water, municipal water and soil
(Colorado State University. Libraries, 2008) Nichols, Tracy A., author; Zabel, Mark, advisor
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy of deer and elk. Research has indicated that CWD is transmitted horizontally, and that both blood and saliva can transmit disease. Environmental exposure to pens where infected animals have been kept has resulted in disease transmission to deer. However, examination of environmental components such as soil and water for prions has been hampered by sensitivity limitations of conventional western blotting and inoculation limitations of bioassays. In this study we evaluated the ability of protein misfolding cyclic amplification (PMCA) to detect protease-resistant prion protein (PrPres) in environmental samples such as water and soil. Serial protein misfolding cyclic amplification (sPMCA) of PrPres, the misfolded proteinase-resistant protein associated with prion disease, was used to detect prion-infected brain homogenate spiked into soil and water to determine detection limits of this assay in environmental samples. The PrPres detection limit for water after 6 rounds of PMCA was 1:26 x 106. Detection of a CWD spike in soil with our current methodology was not possible. We next evaluated surface and drinking water from a CWD endemic region of Colorado for PrPres by sPMCA. PrPres was detected in Cache la Poudre River and flocculant samples at a time of high snowmelt runoff, suggesting that sPMCA can be a useful tool in evaluating water for PrP res in CWD-endemic areas.
Open Access
Human T Lymphotropic Virus Type 1 protein Tax reduces histone levels
(Colorado State University. Libraries, 2008) Bogenberger, James M., author; Laybourn, Pau, advisor
Human T Lymphotropic Virus Type 1 (HTLV-1) is an oncogenic retrovirus that causes adult T-cell leukemia/lymphoma (ATLL). The virally encoded Tax protein is thought to be necessary and sufficient for T-cell leukemogenesis. Tax promotes cell proliferation, represses multiple DNA repair mechanisms, deregulates cell cycle checkpoints, inhibits apoptosis and induces genomic instability. All of these effects of Tax are thought to cooperate in the development of ATLL. In this study, we demonstrate that histone protein levels are reduced in HTLV-1 infected T-cell lines as compared to uninfected T-cell lines, while the relative amount of DNA per haploid complement is unaffected. In addition, we show that replication-dependent histone transcript levels are reduced in HTLV-1 infected T-cell lines, relative to uninfected T-cell lines. Furthermore, we show that Tax expression is sufficient for reduction of replication-dependent histone transcript levels. These results demonstrate that Tax disrupts the proper regulation of replication-dependent histone gene expression. Further, our findings suggest that HTLV-1 infection uncouples replication-dependent histone gene expression and DNA replication, allowing the depletion of histone proteins with cell division. Histone proteins are involved in the regulation of all metabolic processes involving DNA including transcription, replication, repair and recombination. This study provides a previously unidentified mechanism by which Tax may directly induce chromosomal instability and deregulate gene expression through reduced histone levels.
Open Access
Characterization of DJ-1 mutation in mouse astrocytes
(Colorado State University. Libraries, 2008) Ashley, Amanda Kathleen, author; Legare, Marie E., advisor; Hanneman, William H., advisor
Mutations in DJ-1 cause early-onset Parkinson's disease (PD), a progressive, irreversible neurodegenerative condition. Currently, the only known cause of PD is mutation of certain genes including DJ-1, however these mutations account for only 5-10% of overall PD cases. The initial studies attempt to discern if expression of VEGF and HIF1α, factors thought to contribute to both PD as well as carcinogenesis were altered as a result of DJ-1 mutation. In fact, VEGF expression decreased in the brain of DJ-1-/- mice, and increased in lung tissue. As PD is a complex, multi-factorial condition, our studies are designed to incorporate mutation of the PD gene DJ-1 in our target cell type, astrocytes, which are exposed to toxic agents. Overall our results indicate that DJ-1-/- astrocytes do not have an exaggerated phenotype compared to DJ-1+/+ counterparts, however subtle alterations in cell function are observed in mitochondrial membrane potential, expression of proinflammatory mediators, as well as intracellular calcium (Ca2+) dynamics. First, DJ-1-/- astrocytes' resting mitochondrial membrane potential is significantly lower than that of DJ-1+/+ cells. Following treatment with 10μg/mL lipopolysaccharide (LPS), expression of COX2, and NOS2 were similar in both genotypes, however expression of TNFα was significantly lower in DJ-1-/- astrocytes. Finally, a delay in return to baseline intracellular Ca2+ levels following treatment with 1μM ATP was observed in DJ-1-/- cells. Interestingly, expression and secretion of TNFα were decreased in our DJ-1-/- astrocytes following LPS exposure, while expression of COX2 and NOS2 were similar. In conclusion, these changes, though modest, indicate basal dysfunction in astrocyte homeostasis induced by mutation of DJ-1. Secretion of TNFα may be the most significant finding, as it may predispose neurons to degeneration due to lack of sufficient protection against early neurotoxic insults that secreted TNFα may provide. These specific indicators are significant because mitochondrial dysfunction, altered neuroinflammation, and reactive gliosis are all implicated in PD. While altering astrocyte cellular function may not be the primary cause of DJ-1-linked PD, it is possible that changes in this cell type may contribute the progression of parkinsonism.
Open Access
DNA repair and sister chromatid exchange
(Colorado State University. Libraries, 2008) Hagelstrom, R. Tanner, author; Bailey, Susan M., advisor; Liber, Howard, advisor
Mitotic recombination that occurs between sister chromatids, known as sister chromatid exchange (SCE), is a common event in mammalian cells; yet very little is understood about SCE. Likewise, the biological relevance of SCE to humans is also unclear. It is generally thought that SCE represents no permanent alteration to genetic information, however, many cancer prone syndromes present elevated levels of SCE and it is not known whether they are a causal factor in cancer progression or simply a symptom of underlying genomic instability. It has also been purposed that SCE occurring in telomeres (T-SCE) may contribute to the aging phenotype seen in progeroid syndromes. Several accelerated aging syndromes, such as progeria, show highly elevated levels of SCE within telomeric regions. The role of DNA repair in SCE regulation and formation is also under investigation. While it has been shown that at least one of the DNA repair pathways, homologous recombination (HR), is likely to be involved in the formation of SCE, it is less clear whether other DNA repair pathways are also involved in either the formation or suppression of SCE. Therefore, the goal of this research has been to better understand how DNA repair pathways can influence SCE frequency, and how SCE relates to cancer progression and aging. This research also examines how the physical location of SCE, whether it be in genomic (G-SCE) or telomeric (T-SCE) DNA, influence which DNA repair pathways are involved. I examined the role of HR by investigating the Werner (WRN), Bloom (BLM), and FANCD2 proteins. I also investigated the role of non-homologous end joining (NHEJ) by examining the DNA-dependent protein kinase (DNA-PKcs), both the Ku70/80 heterodimer and the catalytic subunit (DNA-PKcs), and Artemis. ERCC1 is a representative member of the final DNA repair pathway examined, nucleotide excision repair (NER). Lastly, I determined if/how DNA repair status can influence the ionizing radiation induced bystander effect (BSE). I was able to determine that at least some of the DNA repair proteins are critical in the generation of a bystander signal providing the first evidence that DNA repair can have an influence via an inter-cellular pathway.
Open Access
Prkdc polymorphisms and radiation effects
(Colorado State University. Libraries, 2008) Askin, Kristin Fabre, author; Ullrich, Robert L., advisor; Weil, Michael M., advisor
The Prkdc gene encodes DNA-PKcs which is involved in the immune system, DNA repair and chromosomal integrity. In humans, deficiencies in DNA-PKcs are linked to cancer predisposition. This connection can also be observed in mice models using ionizing radiation as the carcinogenic inducer. In particular, the BALB/c mouse strain is susceptible to mammary cancer after radiation exposure. Subsequent studies have shown that DNA repair is deficient in BALB/c which is attributed to a hypomorphic variant of DNA-PKcs. DNA-PKcs has also been linked to apoptosis because BALB/c is more resistance to ionizing-induced apoptosis in intestinal crypt cells compared to other mouse strains. Furthermore, it has been demonstrated that DNA-PKcs is involved in telomere maintenance. Additional studies have revealed two polymorphisms in Prkdc BALB/c that may be responsible for its DNA-PKcs variant.
Open Access
Chromosomal aberrations in the tumor and peripheral blood and changes in aberrations during treatment of canine lymphoma
(Colorado State University. Libraries, 2008) Devitt, Jennifer J., author; LaRue, Susan M., advisor
Lymphoma is the most frequently diagnosed hematopoietic malignancy in dogs. Untreated, the survival times are approximately one month. Chemotherapy is the current standard of care and can initiate and temporarily maintain remission, with average survival times of one year. Cytogenetic abnormalities can aid in diagnosing tumors as well as in giving a more accurate prognosis for the specific mutations present. In human lymphoma patients, chromosomal changes from peripheral lymphocytes have been used prognostically and to document response to treatment. Evaluating peripheral lymphocytes instead of tumor cells is less invasive for the patient and technically easier. Recurrent aberrations have been reported in canine lymphomas. Since this cancer parallels human Non-Hodgkin's Lymphoma which has recurrent chromosomal anomalies that have been correlated with clinical behavior of the tumor and patient survival, it reasons that canine lymphoma would as well. This study was designed to investigate a correspondence between numerical aberrations detected in the tumor and the peripheral blood in dogs with lymphoma. Additionally, the peripheral blood aberrations were monitored during the course of treatment to document changes seen during remission and at the time of disease recurrence. Twenty-five dogs with lymphoma had one lymph node excised, a peripheral blood sample drawn, and a bone marrow aspirate performed. A portion of the lymph node was submitted for histopathology and immunophenotyping and another portion was retained for cytogenetic analysis. The peripheral blood sample was cultured for chromosome counting and cytogenetic analysis. The bone marrow aspirate was used for staging purposes. A significant correspondence between the numerical aberrations in the tumor and the peripheral blood was found with six out of the seven numerical aberrations demonstrating predictive value of the peripheral blood. During the course of treatment, the quality and quantity of aberrations changed, likely due to DNA damaging treatment modalities. Once treatment ended, the frequency of aberrations diminished. A prognostic significance could not be determined using the additional diagnostic information that was garnered such as age, gender, histological classification, breed, immunophenotype, or stage of disease. This was probably due to a limited sample size and is worthy of further investigation.
Open Access
The role of Cdc42, ADF/cofilin, myosin II and waves during the establishment of neuronal polarity
(Colorado State University. Libraries, 2008) Flynn, Kevin Carl, author; Bamburg, James R., advisor
The establishment of neuronal polarity is an essential developmental process, underlying the unidirectional flow of information in neurons and the overall function of the nervous system. In cultured hippocampal neurons, the first signs of polarity occur as one of several undifferentiated processes begins to elongate rapidly to form the axon (axonogenesis). The regulation of the cytoskeleton and intracellular trafficking are crucial to the proper development of neuronal polarity. This dissertation explores both of these polarity-developing mechanisms, identifying actin-regulating components in the signaling pathways as well as characterizing growth-cone like "waves" that correlate with axonogenesis. This study begins by analyzing the functional consequences of the loss of the Rho GTPase, cdc42, on the polarization of hippocampal neurons. Neurons from the cdc42 knock-out (cdc42KO) mouse have severe deficiencies in their ability to extend axons in vivo and in culture which is exerted through the regulation of actin dynamics. The actin regulating protein, cofilin is normally asymmetrically enriched in its active form in axonal growth cones but in cdc42 KO neurons there is an increase in the phosphorylation (inactivation) of cofilin. Cofilin expression promotes axon growth, whereas cofilin knockdown results in polarity defects analogous to those seen upon cdc42 ablation. Taken together, these data suggest that cdc42 is a key regulator of axon specification and that cofilin is a downstream effector of during this process. Though these studies suggest the involvement of cofilin downstream of cdc42; active cofilin cannot rescue polarity deficits in cdc42KO neurons. This suggests that other actin regulating proteins may also be required for axon formation. The actin motor protein, myosin-II also shows an increased activation in the cdc42KO brain. Inhibition of myosin II activity promotes axon formation and acts in synergy with cofilin on inducing supernumerary axons. Furthermore, the combined inhibition of myosin and activation of cofilin rescues axon formation in cdc42KO neurons while either treatment individually does not. Thus, the concurrent inhibition of myosin and activation of cofilin can contribute to the regulation of actin dynamics during axon specification. Axon specification is dependent on the transport of materials to the developing axonal growth cone. "Waves"-growth cone-like structures, propagate down neurites and correlate with neurite extension; thus, waves have been suggested as a mechanism for transporting materials that support this growth. Waves occur in all processes during early neuronal development, but are more frequent in the developing axon. Proteins enriched in axonal growth cones are also localized to waves and proteins such as cofilin and actin appears to be transported via waves to the growth cone, suggesting that waves represent a transport mechanism. Wave arrival at neurite tips was also coincident with an increase in growth cone size and dynamics. In addition, waves can promote neurite branching, either by supporting the growth of existing branches or by facilitating the growth of nascent branches. Waves are observed in neurons in organotypic hippocampal slices, a 3-dimensional growth environment reflecting the in vivo environment. Together, these data indicate that waves contribute to axon differentiation and growth both through the transport of actin and by increasing growth cone dynamics.
Open Access
Ovine pulmonary adenocarcinoma as an animal model for human lung adenocarcinoma
(Colorado State University. Libraries, 2008) Hudachek, Susan, author; Dernell, William S., advisor
Appropriate animal models of disease allow defined and controlled investigations that can ultimately be applied to the management of human disease. Based on symptomatic, histopathologic, and possible molecular signaling similarities, we hypothesized that sheep experimentally affected by OPA are a relevant animal model for the study of human lung adenocarcinoma and, in particular, for the evaluation of lung cancer therapeutics. The value of this model is dependent upon its predictability, reproducibility, amenability, and validity. The former two features have been previously reported; OPA induction in sheep is both predictable and reproducible following JSRV inoculation of neonatal lambs. The overall objective of this body of work was to assess the amenability of this animal model for therapeutic research and to assess the validity of OPA-affected sheep as an animal model for human lung adenocarcinoma in terms of genetic similarities. We determined that this animal model is amenable for therapeutic studies because, using CT, OPA can be detected early, before the onset of clinical signs, and cancer development can be monitored noninvasively. However, not only did we observe OPA disease progression during this study, but surprisingly, we also witnessed spontaneous regression of OPA. In fact, the latter was the more common outcome seen in our research after JSRV inoculation of neonatal lambs. We propose that the immune system, particularly CD3+ T-cells, is an important mediator of the spontaneous regression of JSRV-induced OPA seen in our work. Regardless of the cause, the mere occurrence of spontaneous regression of cancer in OPA-affected sheep severely restricts the use of this animal model for therapeutic research. In addition to assessing the amenability of OPA-affected sheep for therapeutic research, we also found that OPA tumors do not harbor genetic mutations in the TK domain of the EGFR, KRAS codons 12 and 13, or the DNA-binding domain of P53 and therefore, are not genetically similar to human lung adenocarcinomas that contain these mutations. Based on these genetic disparities, OPA-affected sheep are not an ideal animal model for human lung adenocarcinoma. Overall, the genetic profile combined with the disease development data provided further characterization of OPA and facilitated an assessment of the utility and relevance of this animal model for human lung cancer studies.
Open Access
Isolation and characterization of proteins that interact with a pollen-specific calmodulin-binding protein
(Colorado State University. Libraries, 2008) Shin, Sung-Bong, author; Reddy, A. S. N., advisor
Calcium and calmodulin, a calcium sensor, are implicated in pollen germination and tube growth. However, the mechanisms by which calcium and calmodulin regulate these processes are largely unknown. Calcium bound calmodulin regulates diverse cellular processes by modulating the activity of other proteins called calmodulin-binding proteins. Maize pollen-specific calmodulin-binding protein (MPCBP) and its homolog (NPG1, no pollen germination) from Arabidopsis were isolated previously. Studies with a knockout mutant have shown that AtNPG1 is not necessary for pollen development but is essential for pollen germination. Analysis of the Arabidopsis genome sequence with AtNPG1 revealed the presence of two other proteins (AtNPGR1, NPG-Related1; AtNPGR2, NPG-Related 2) that are closely related to AtNPG1. To gain insights into the function of AtNPG1 and AtNPGRs, I focused my research on characterization of these proteins. Specifically, my research focused on in vivo localization of AtNPG1 in pollen grain and tube, interaction between AtNPGs, isolation and characterization of AtNPG1 interacting proteins, and functional analysis of AtNPGR1 in plant development. Transgenic plants containing GFP fused to AtNPG1 promoter showed GFP expression only in mature and germinating pollen, suggesting that the promoter is active only in pollen. Localization of GFP-AtNPG1, driven by AtNPG1 promoter, during different stages of pollen germination revealed uniform cytosolic distribution of GFP-AtNPG1 in the growing pollen tube that was similar to GFP alone. However, the observed uniform localization of GFP-AtNPG1 is not due to degraded fusion protein. AtNPGRs, like AtNPG1, bind calmodulin in a calcium-dependent manner. The calmodulin-binding domain in AtNPGs was mapped to a short region. AtNPG1 and AtNPGRs have several tetratricopeptide repeats (TPRs) that are known to be involved in protein-protein interaction. I tested the interaction among AtNPGs using the yeast two-hybrid analysis. AtNPG1-BD interacted with itself-AD and AtNPGR1-AD and AtNPGR2-AD. AtNPGR1-BD interacted with itself-AD, AtNPG1-AD and AtNPGR2-AD. However, AtNPGR2-BD did not interact with AtNPG1-AD or AtNPGR1-AD and showed a very weak interaction with itself-AD. To study the role of AtNPG1, AtNPG1 interacting proteins from a petunia pollen library were isolated in a yeast two-hybrid screen and identified as pectate lyase-like proteins. Using in vivo and in vitro protein-protein interaction assays, I show that AtNPGs interacts with four Arabidopsis pectate lyase-like (PLL) proteins with the highest similarity to petunia PLLs. Truncated AtNPG1 lacking the TPR 1 did not interact with most of partners or showed drastically decreased interaction with some proteins, suggesting that the TPR 1 domain is essential for this interaction. To understand the role of Arabidopsis PLL proteins, we characterized these using molecular and biochemical tools. Of the 26 Arabidopsis PLLs, fourteen were expressed in pollen and four AtPLLs were highly expressed. These four AtPLLs showed expression in other tissues also. Analysis of pectate lyase activity in Arabidopsis tissues (flower, root, stem, and leaf) revealed enzyme activity in all four tissues and the activity varied depending on the buffer pH. To see if AtNPG1 interacting AtPLLs have enzyme activity, four AtPLLs were expressed in bacteria or yeast and assayed for their enzyme activity under different conditions with different substrates. None of the AtPLLs expressed by bacterial or yeast showed pectate lyase activity. To discover the role of AtPLL in Arabidopsis development, one AtPLL mutant, atpll8, was isolated. Phenotypic analysis of atpll8 under different growth condition showed no significant differences as compared to wild type. AtNPGR1, unlike AtNPG1, is expressed in tissues other than pollen. To understand the role of AtNPGR1 in plant development, I isolated an atnpgr1 knockout mutant and characterized its phenotype under different growth conditions. The atnpgr1 showed a sugar resistance phenotype, suggesting that it might be involved in sugar sensing and/or signaling pathway. Expression of hexokinase (Hxk), an important component in sugar signaling in plants, and other genes in the Hxk pathway, revealed that NPGR1 might be involved in an Hxk independent pathway.
Open Access
Examination of molecular genetic factors involved in sensitivity to breast cancer following radiation exposure
(Colorado State University. Libraries, 2008) Williams, Abby J., author; Ullrich, Robert L., advisor; Bailey, Susan M., advisor
Understanding DNA repair is not only an important aspect of cell biology, but also has important implications for the field of carcinogenesis since cancer most likely occurs from genetic damage that occurs over one's lifetime. DNA repair needs to be accurate and efficient in order for a cell to maintain genomic stability, and defects in repair systems can result in radiosensitivity. Because radiation exposure, DNA repair deficiency and telomere malfunction are associated with cancer risk, we investigated Lymphoblastoid Cell Lines (LCLs) from breast cancer patients and controls for chromosomal radiosensitivity, relative telomere length, and gene expression changes. The importance of studying peripheral blood lymphocytes from cancer patients lies in the fact that minimally invasive techniques are lacking for the detection of individuals with high risk for cancer, and that telomere length has been proposed to be useful in this regard. Identification of radiosensitivity markers would be a valuable contribution for clinicians in hopes of avoiding excessive radiation or chemotherapy treatment given to patients. Failure to adequately repair DNA damage can result in cell suicide or halting of cell cycle progression in an attempt to allow repair mechanisms to operate. If damage persists, a cell can be pushed toward transformation and the pathway of carcinogenesis. A second aspect of the current work was to study the Homologous Recombination double-strand break repair protein, Rad51D. The emerging interrelations between DNA repair and telomere maintenance also prompted us to evaluate Rad51D's role in telomere function. The final aspect of this research involved examination of how DNA repair related proteins are linked to the indirect effect of ionizing radiation exposure known as the bystander effect (BSE). We are the first to demonstrate that DNA-PKcs and ATM are required to generate, but not receive, a bystander signal. We also show that mouse embryonic fibroblasts do not generate bystander signals to neighboring cells, while their adult cell counterparts do. Taken together, this work makes important contributions to our appreciation of the many and varied roles DNA repair related proteins play in maintenance of chromosomal integrity, proper telomere function, inhibition of carcinogenesis and now, regulation of the BSE.
Open Access
Interactions between plants and an opportunistic human pathogen, Pseudomonas aeruginosa
(Colorado State University. Libraries, 2008) Weir, Tiffany L., author; Schweizer, Herbert P., advisor; Vivanco, Jorge M., advisor
Pseudomonas aeruginosa is an opportunistic human pathogen that can be found living in soil, water, or saprophytically on plant tissues. It is important to understand the pathology of this organism under variable conditions because of its ability to survive in diverse environments, its role in human disease, and its use as a model organism in studies on biofilm formation, quorum-sensing, and pathogenicity. To this end, a number of unconventional model systems, including plants, nematodes, and fruit flies, have been developed to study the pathology of P. aeruginosa. In the present study, the interactions between P. aeruginosa and plants, with respect to pathogenicity, quorum-sensing, and microbial ecology are further explored. To examine what factors are important in the pathogenicity of P. aeruginosa in a plant system, compatible and incompatible cultivars of Nicotiana tabacum were infiltrated with the pathogen. Bacterial growth in planta was monitored and P. aeruginosa PAO1 gene expression was examined 24 hours after infiltration into the hosts. The data suggests that, in addition to known virulence factors, the acquisition of micronutrients such as sulfate and inorganic phosphate are also important in disease development. The results of this study also suggest that type III secretion systems may be important in P. aeruginosa's ability to infect plants, and that differences in host response, i.e. salicylic acid signaling, are determining factors in host compatibility. Another aspect of this study was to utilize the natural interactions between plant roots and soil-borne bacteria to identify root exudates that interfere with bacterial quorum sensing (QS), particularly in P. aeruginosa. Quorum sensing in P. aeruginosa controls the expression of several secreted factors that are important in virulence of the pathogen, and preventing infections by inhibition of quorum sensing is a current therapeutic target. Unfortunately, while many of the exudates appeared to have some affect on QS in general, none had strong activity against P. aeruginosa QS systems. However, one class of chemicals, triterpene saponins, was shown to be active in a lux-based QS reporter. Finally, preliminary data suggesting that root exudates can influence competitive outcomes between two soil-borne bacterial species are also presented. The interactions between bacteria are typically studied in nutrient rich medium under defined laboratory conditions. Under these conditions, P. aeruginosa outcompetes Agrobacterium tumefaciens, two bacteria that potentially compete for the same niche in the soil. However, when Arabidopsis thaliana is factored into this equation, growth of A. tumefaciens is favored. Furthermore, the negative effects of P. aeruginosa on the growth of A. thaliana were reduced.
Open Access
Comparison of radiobiological endpoints in cells from CXB RI mice
(Colorado State University. Libraries, 2008) Xiao, Guanxiong, author; Weil, Michael M., advisor
Recombinant inbred (RI) mouse strains have been used both for trait cosegregation studies and genetic linkage analysis. They are created by using a breeding scheme that consists of a cross between two inbred mouse strains (progenitor strains) followed by at least 20 generations of brother-sister inbreeding. Thus, RI strains are inbred (homozygous at every locus) and derive roughly half their genome from each of the two progenitor strains. The CXB RI strain set consists of 13 RI strains derived from matings of BALB/c (C) and C57BL/6(B) mice. The CXB progenitor strains, BALB/c and C57BL/6, differ in their susceptibility to radiation-induced mammary tumors with BALB/c being susceptible and C57BL/6 being resistant. In part, the susceptibility difference can be explained by a polymorphism in the Prkdc gene which encodes the catalytic subunit of DNA-dependent protein kinase. However, other, as yet unknown, loci may be involved. The CXB RI strain set provides a useful tool to unravel the events that lead to radiation-induced mammary tumorigenesis and to understand the interrelationships of cellular radiobiological endpoints to one-another. We have generated fibroblast strains from each of the CXB RI strains and from the progenitor strains. The fibroblast strains were assayed for a number of radiobiological endpoints including clonogenic survival following acute and low dose-rate exposures, γ-H2AX focus formation and clearance following acute and low dose-rate exposures, and G2 chromosomal aberrations. In addition, we genotyped the strains for a polymorphism in the gene encoding the catalytic subunit of the DNA-dependent protein kinase, Prkdc. We then determined the correlations of different endpoints between the RI strains. As expected, clonogenic survival at low dose rates and following acute exposures were positively correlated. γ-H2AX focus formation at low dose rate correlated well with survival endpoints, particularly clonogenic survival under low dose rate irradiation and the surviving fraction at 2 Gy acute exposures. These three endpoints are all significantly associated with the Prkdc genotype with radiosensitive strains having the BALB/c genotype. The data we have collected provides a baseline description of cellular radio sensitivity in CXB fibroblasts. The approach used in this dissertation can be used to correlate these cellular radiobiological endpoints with susceptibility to clinically significant adverse outcomes from cancer radiotherapy, such as normal tissue injury and radiation-induced second cancers.
Open Access
Glial inflammatory signaling in manganese neurotoxicity
(Colorado State University. Libraries, 2009) Moreno, Julie Ann, author; Tjalkens, Ronald B., advisor
Degenerative movement disorders affecting the basal ganglia, including, Parkinson's (PD) and Huntington's diseases, are debilitating and currently incurable. Increased inflammatory gene expression in astrocytes promotes neuronal loss in these disorders, but the signaling mechanisms underlying this phenotype are not fully understood. In order to enhance understanding of this phenotype, the degenerative movement disorder, manganism, is a useful model, because patients suffering from excessive exposure to manganese (Mn) develop a neurodegenerative condition affecting the same brain region and with clinical features resembling PD. Recently, the potential effects of Mn on the developing brain have gained attention due to an increase in cognitive deficits with overexposure to Mn. Moreover, astrocytes are a known target of Mn, and reactive gliosis seems to precede neuronal injury. Mn toxicity enhances production of the inflammatory mediator nitric oxide (NO) in astrocytes by a mechanism involving NF-κB, the principal transcription factor responsible for expression of inducible nitric oxide synthase (NOS2). However, the role Mn toxicity plays in the developing brain along with the signaling mechanism(s) by which Mn enhances activation of NF-κB remains poorly understood in astrocytes. Therefore, in order to address the gap in knowledge I have characterized the role of glial cells in the promotion of neuronal damage in the developing and adult brain in a mouse model of Mn neurotoxicity, as well as the mechanism by which Mn enhances inflammatory activation of NF-κB dependent genes in astrocytes. First, it was identified that sGC relays signals to ERK and NF-κB, initiating NO signaling in astrocytes. Also it was determined that the glial inflammatory response leads to an age- and sex-dependent vulnerability of the basal ganglia which can be modulated by E2. This indicates that Mn toxicity in the developing brain results in locomotor deficits, reduction in normal dopaminergic neurotransmitter release, increased NOS2 expression in glial cells and neuronal injury. These findings are significant because once the mechanism of Mn-induced inflammatory activation of glial cells is understood, it will promote a better understanding of manganism and potentially other disorders of the basal ganglia.
Open Access
An evaluation of biological responses to model biomaterials in vivo and in vitro
(Colorado State University. Libraries, 2009) Chamberlain, Lisa M., author; Gonzalez-Juarrero, Mercedes, advisor
The use of in vitro and in vivo models to study inflammatory responses is extremely common in the pre-clinical evaluation of implantable materials and anti-inflammatory drugs. In this body of work we performed comparative studies of the inflammatory responses elicited by different biomaterials when interacting with cells in the implanted host. The results demonstrate first the non-equivalence between immortalized cell lines and primary-derived cell types in the inflammatory response and second temporal effects on differences in responses. Additionally, differences between in vivo and in vitro models are clearly demonstrated, and potential differences between our in vivo and other published models are seen. The results obtained from this comparative study will help to explain many discrepancies found between previous studies reported in the literature.
Open Access
The effects of mannose capped lipoarabinomannan on dendritic cell function
(Colorado State University. Libraries, 2009) Lee, Eric John, author; Gonzalez-Juarrero, Mercedes, advisor; Schenkel, Alan, committee member; Chen, Chaoping, committee member
M. tuberculosis is one of the leading causes of death due to infectious disease in the world. While the majority of people are capable of controlling the initial infection, many progress to a latent stage of disease where the M. tuberculosis bacilli persist for long periods of time within the host. The M. tuberculosis cell wall lipoglycan mannose capped lipoarabinomannan (ManLAM) has been characterized as one of the immunomodulatory factors associated with the bacteria [3-5]. ManLAM interacts with dendritic cells (DCs) via DC-SIGN, mannose receptors and to a lesser extent TLR-2 [6-8]. Thus we set out to examine the effects that ManLAM has on DCs both in vitro and in vivo. ManLAM treatment of bone marrow derived DCs (BMDCs) prevents their phenotypic maturation reduces the expression of MHC class II and CD1d. BMDCs stimulated with ManLAM also exhibit altered phagocytic capacity and the inability to stimulate naïve CD4+ T-cell proliferation.
Open Access
Characterization of the flow cytometry mutation assay and its use in novel genotoxicity studies
(Colorado State University. Libraries, 2009) Keysar, Stephen Berkeley, author; Fox, Michael H., advisor
The flow cytometry mutation assay (FCMA) has been previously demonstrated to be a rapid and sensitive assay for measuring mutations induced by a wide variety of genotoxic agents. After treatment with a mutagen, the mutant fraction measured by the FCMA increased to a peak over time and subsequently decreased to a stable plateau. Using ethyl methanesulfonate (EMS), ionizing radiation (IR) and asbestos, I determined that the return to normal cell survival is an indicator of peak mutant expression. Decreased survival significantly contributes to the decline in the mutant fraction and confirms that mutations that are not clonogenically viable are measured by the FCMA on the peak day of expression. Also, I analyzed clones isolated from several CD59- regions and generated mutant spectra for EMS using flow cytometry. I then investigated hypoxia induced mutagenesis. It has been previously shown that hypoxic stress can generate DNA damage and mutations which are likely caused by reactive oxygen species (ROS). Here I demonstrate that the oxygen radical scavenger dimethyl sulfoxide significantly decreased cell killing and mutagenesis after hypoxia treatment, supporting the concept that ROS are responsible for hypoxia induced mutations. I also investigated the effects of silencing of DNA repair proteins on cell survival, cell cycle and mutagenesis. The knockdown of homologous recombination repair protein Rad51C slightly increased sensitivity to IR and drastically increased killing by EMS treatment. Rad51C knockdown also caused a significant G2 phase buildup after EMS treatment. Silencing of the non-homologous end joining (NHEJ) protein Ku80 increased cell sensitivity to IR and decreased the mutant yield after EMS treatment. This implies that EMS generates significant double strand breaks (DSBs) during S phase that are possibly repaired by NHEJ. In summary, I have demonstrated that the FCMA is a fast and reliable method to measure mutagenesis induced by an agent and to quantify the degree of damage by obtaining a mutant spectrum. I have used this assay to investigate mechanisms of mutagenesis by EMS, IR, asbestos and hypoxia and evaluated the effects of DNA repair pathways on cell cycle, cell killing and mutant induction.