Theses and Dissertations

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Open Access
A cellular prion protein-dependent signaling pathway for proinflammatory cytokine- and β-amyloid-induced cofilin-actin rod formation
(Colorado State University. Libraries, 2014) Walsh, Keifer P., author; Bamburg, James, advisor; Zabel, Mark, committee member; Tjalkens, Ron, committee member
Stimulus of oxidative stress in neurodegeneration leads to synaptic dysfunction and the eventual loss of neurons in the central nervous system. The actin cytoskeleton of neurons under acute or chronic stress experiences dynamic remodeling due to functional alterations in the actin depolymerizing factor (ADF)/cofilin family of actin-binding proteins. Once oxidized, disulfide cross-linked cofilin incorporates into the formation of tandem arrays of 1:1 cofilin:actin rod-like bundles (rods). Rods sequester cofilin, which is required for synaptic remodeling associated with learning and memory, and interrupt vesicular transport by occluding the neurite within which they form. Different rod-inducing stimuli target distinct neuronal populations within the hippocampus. Rods form rapidly (5-30 min) in >80% of cultured hippocampal neurons which are treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% maximum response in ~6 h) occurs in ~20% of neurons upon exposure to soluble beta-amyoid dimer/trimer (Aβd/t), a physiologically relevant species in Alzheimer disease (AD). Here we show that proinflammatory cytokines (TNFα, IL-1β, IL-6) induce rods at the same rate and in the same subpopulation of hippocampal neurons that respond to Aβd/t. Rod formation by proinflammatory cytokines may link the neuroinflammatory hypothesis for AD with the Aβ hypothesis by providing a common target. Neurons from PrPC-null mice form rods in response to glutamate or antimycin A, but not in response to Aβd/t or proinflammatory cytokines. Prion-dependent rod inducers require the activation of NADPH oxidase (NOX) to generate reactive oxygen species (ROS), but NOX activity is not required for rods induced by glutamate or energy depletion. Aβd/t and TNFα stimulate cofilin dephosphorylation and increased ROS production in a subpopulation of neurites at levels that exceed a minimum threshold to maintain stable rods. Removing inducers or inhibiting NOX activity in cells containing prion-dependent rods causes rod disappearance with a half-life of ~36 minutes. Interestingly, the overexpression of PrPC alone is sufficient to induce rods in >40% of hippocampal neurons, nearly twice the number that respond to Aβd/t or TNFα. This suggests that membrane microdomains containing PrPC recruit the oxidizing machinery necessary to initiate and sustain rod formation. Our hypothesis is supported by the inhibition and reversal of prion-dependent rods by the naturally occurring plant triterpene, ursolic acid (UA), and the pharmacological peptide RAP310. UA and related compounds to RAP310 have been proposed to inhibit changes in the membrane lipid profile that permit LR coalescence. The vast majority of neurodegenerative disorders are considered sporadic in incidence and multifactorial in cause, making treatment at an early stage a significant challenge. If cofilin-actin rods indeed bridge multiple disease initiating mechanisms into a common pathway leading to synapse loss, they provide a valuable target for therapeutic intervention.
Open Access
A metabolomics approach for examining synbiotic protection against infectious enteric pathogens
(Colorado State University. Libraries, 2019) Nealon, Nora Jean, author; Ryan, Elizabeth P., advisor; Dean, Gregg, committee member; Henry, Charles, committee member; Tobet, Stuart, committee member
Infectious gastrointestinal diseases contribute to billions of global cases of human illness annually. Salmonella enterica serovar Typhimurium and human rotavirus represent two human health challenges, where escalating multidrug resistance and poor vaccine efficacy warrant the development of alternative treatments. Health-promoting probiotic microorganisms are becoming increasingly studied for their production of bioactive small molecules that confer protective effects against enteric pathogens. Among probiotics, Lactobacilli, Bifidobacteria and E. coli Nissle form synbiotics with rice bran, the prebiotic-rich outer coating of brown rice, to enhance animal protection against S. Typhimurium infection and human rotavirus diarrhea compared to probiotics or rice bran alone. Despite these beneficial interactions of probiotics and rice bran, a knowledge gap exists in our understanding of the synbiotic small molecules driving these protective effects, especially across probiotic species differences in small molecule production. To test our overarching hypothesis that probiotic species would metabolize rice bran into distinct suites of small molecules that suppressed pathogen function, we first applied the cell-free supernatant from L. paracasei, L. fermentum, and L. rhamnosus cultured with rice bran to S. Typhimurium and observed magnitude-dependent growth suppression across synbiotics. Both L. paracasei and L. fermentum supernatants exhibited enhanced growth suppression compared to their probiotic-only treatments and contained differentially abundant antimicrobial lipids, amino acids, and nucleotides that have not been previously characterized for antimicrobial functions. The cell-free supernatant of the L. paracasei and L. fermentum synbiotics were fractionated and applied to S. Typhimurium to identify the small molecules driving their enhanced Salmonella growth suppression. Metabolite profiles were also compared across synbiotics. Each synbiotic produced several bioactive fractions that suppressed Salmonella growth. While both L. fermentum and L. paracasei bioactive fractions contained abundant lipids, L. fermentum fractions were selectively-enriched in the energy metabolite fumarate and L. paracasei fractions were uniquely-enriched with amino acids (imidazole lactate, ornithine) suggesting that Lactobacillus spp. probiotics could differentially metabolize rice bran to drive Salmonella growth suppression with different suites of small molecules. To examine probiotic metabolism of rice bran in mammalian systems, we compared the intestinal and blood metabolomes of healthy adult mice and gnotobiotic, neonatal pigs that were fed combinations of probiotics and rice bran to the metabolomes of animals consuming rice bran or probiotics alone. In mice, a notable difference following 15 weeks consumption of B. longum fermented was that the arginine metabolite N-delta-acetylornithine was significantly increased in B. longum fermented rice bran compared to rice bran alone and was elevated in both the colon tissue and blood of mice consuming fermented rice bran compared to rice bran alone. In gnotobiotic neonatal pigs, three weeks of prophylactic supplementation with E. coli Nissle and L. rhamnosus GG and rice bran were more effective at reducing human rotavirus diarrhea compared to pigs given these probiotics or rice bran alone. Approximately 300 colon and blood metabolites that were differentially-abundant between synbiotic-consuming pigs versus pigs consuming probiotics alone were identified, over 50% of which were lipids and amino acids. Similar modulations lipid and amino acid metabolites (sphingolipids, diacylglycerols, arginine metabolites) were identified in the colon tissue and blood of mice and pigs consuming the synbiotic treatments. Consequently, the association of these metabolite profiles with human rotavirus diarrhea protection, when combined with their presence in two mammalian models, provides strong rationale for these infectious enteric disease protective roles harbored by these metabolites. The results of these studies provide a role for synbiotics in the prevention of infectious gastrointestinal diseases. For the first time, high-throughput metabolomics analyses were applied to identify differential bioactive metabolite production by Lactobacillus spp. + rice bran synbiotics that suppressed S. Typhimurium growth, as well as to compare bioactive metabolites produced by B. longum, L. rhamnosus GG, and E. coli Nissle in mice and pigs that were protective against human rotavirus diarrhea. The contributions of amino acids and lipids to the enhanced capacities of these synbiotics compared to probiotics or rice bran alone can be studied further for their mechanisms of action on pathogens. Ultimately, these bioactive synbiotic metabolites can guide the optimization and development of broad-spectrum antimicrobials and other prophylactic agents that protect against infectious enteric diseases across the human and animal lifespan.
Open Access
Actin dynamics in silico, in waves, and in rods
(Colorado State University. Libraries, 2009) Pak, Chi W., author; Bamburg, James, advisor
The current paradigm of actin dynamics and superorganization has advanced in the past decade from emerging technologies and perspectives, which include the discovery of actin nucleators, real time imaging of the dynamics of single filaments in vitro, and single molecule imaging of actin superstructures in vivo. These advances have influenced each of our studies on multiple levels, sometimes directly. A novel analysis of single actin filament dynamics revealed faster than expected dynamics during treadmilling but not during bulk polymerization. Using an exact stochastic simulation, we investigated whether filament-annealing and -fragmentation might account for faster than expected dynamics; their influence on actin dynamics had not been investigated before in a comprehensive model. Results from our work demonstrated that filament-annealing and -fragmentation alone cannot account for faster than expected dynamics during treadmilling. Thus, strictly through computational modeling, we are able to investigate various hypothetical models and offer insights into a process that cannot be achieved by experimentation. A concept that has also gained support during the past decade has been the self-organizing nature of actin, which was demonstrated by the Listeria actin-comet-tail reconstitution assay. We have proposed that this is a fundamental property of all actin superstructures, whether they are assembled in vitro or in vivo or whether they are involved in development or disease. The concept of actin's self-organization has influenced our study of neuronal waves, which are growth cone-like structures that travel along neurites and which were hypothesized to transport actin to growth cones and support neuritogenesis. Using diffusional analysis, we were able to demonstrate that neuronal waves transport actin. Neuronal waves provide a unique mechanism for transporting actin in that the delivery of actin is dependent upon actin itself and its dynamics. In disease states, the self-organization of actin is often changed but not disrupted, sometimes resulting in the formation of orderly-structured aggregates of cofilin and actin known as cofilin-actin rods (or rods). Using glutamate excitotoxicity as a model system for the cofilin pathology observed in Alzheimer disease (AD), we have determined signaling mechanisms for cofilin-actin rod induction, which in young rat hippocampal neurons require AMPA receptors and are calcium-independent. In addition, cofilin-actin rod interactions with microtubule associated proteins, and associated changes to the microtubule cytoskeleton were studied for its potential relevance to the pathology of AD. Our results suggest that disruptions to the normal organization of actin and microtubules might underlie several pathological hallmarks of early AD.
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
An investigation of the molecular complexities that regulate molting in decapod crustaceans
(Colorado State University. Libraries, 2015) Pitts, Natalie Lynn, author; Mykles, Donald L., advisor; Garrity, Deborah M., committee member; Tjalkens, Ronald B., committee member; Tsunoda, Susan, committee member
Molting in decapod crustaceans is regulated by the interaction of two hormones, molt inhibiting hormone (MIH) and ecdysteroids. Ecdysteroids are steroid hormones secreted from the molting gland or Y-organ (YO) and fluctuations in hemolymph ecdysteroid titers regulate progression through the molt cycle. Secretion of ecdysteroids is controlled by the peptide hormone MIH, which is synthesized and released from the X-organ/sinus gland (XO/SG) complex in the eyestalk ganglia (ESG). The field of crustacean endocrinology has mainly focused on understanding the molecular underpinnings of MIH’s action on ecdysteroid production in the YO. The goal of this dissertation was to examine how MIH synthesis and secretion from the XO/SG complex contributes to molt cycle progression. Blackback land crabs, Gecarcinus lateralis, were induced to molt via autotomy of five or more walking legs (multiple limb autotomy or MLA). ESG were collected from intermolt, premolt, and post-molt animals and changes in expression of Gl-MIH and mTOR signaling pathway components were investigated. There was a significant effect of molt stage on Gl-MIH and mTOR signaling pathway gene expression in the ESG of G. lateralis. Continuous elevation of MIH transcript abundance during pre and post molt indicates that MIH titers in the hemolymph are not regulated by changes in transcript abundance. Molting also significantly increased expression of Gl-Akt, Gl-mTOR, Gl-Rheb, and Gl-S6K in one or more molt stages. Akt inhibits the tuberous sclerosis complex allowing for the activation of Rheb. Rheb is a GTPase that binds and activates the mechanistic target of rapamycin (mTOR). mTOR activates S6 kinase (S6K), increasing protein synthesis. ESG of naturally molting green crabs, Carcinus maenas, were also collected from intermolt, early premolt, and post molt animals. Molting had little effect on gene expression in C. maenas, confirming previous findings that molt progression is regulated post transcriptionally. This dissertation identifies a novel nitric oxide (NO) binding protein in the SG of C. maenas. The hypothesis is that NO negatively regulates MIH secretion from the SG thereby controlling molt progression. This unidentified endogenous binding protein allows NO to be present in the SG for a prolonged period and can therefore continually regulate neuropeptide release. Localization of the enzyme that produces NO (nitric oxide synthase; NOS) and MIH in the SG of C. maenas, G. lateralis, and Metacarcinus magister is consistent with the hypothesis that NO is a regulator of neuropeptide release in the crustacean SG. The second goal of this dissertation was to explore why some crustacean populations or individuals within a population are refractory to molt induction. The Bodega Bay population of C. maenas is refractory to molt induction techniques and a similar phenomenon is observed in G. lateralis. Some G. lateralis induced to molt via MLA did not enter premolt 90 days post induction and were classified as "blocked." These animals underwent a second molt induction technique, eyestalk ablation (ESA), and YO, brain (Br), and thoracic ganglia (TG) were collected at 1, 3, and 7 days post ESA. Gene expression of MIH and mTOR signaling pathway genes was examined in all three tissues (see Figure 3 for MIH signaling pathway components and there interactions). Results from this experiment suggested that a similar mechanism of molt resistance exists between C. maenas and G. lateralis. ESA did not increase hemolymph ecdysteroid titers of blocked animals, whereas ESA significantly increased ecdysteroid titers in control and intermolt animals. Gl-MIH expression in the ESG and expression of many Gl-MIH signaling components in the YO were upregulated in blocked animals, suggesting that the blocked animals were in a "hyper-repressed" state, and therefore resistant to molt induction by ESA and MLA. In both species, MIH is expressed in the Br and TG. The hypothesis is that MIH secretion from these other central nervous system (CNS) tissues contributes to a resistance to molt induction techniques. Expression of MIH signaling pathway genes is unchanged in the Br and TG in response to ESA. These data suggest that MIH does not activate a signaling pathways in CNS tissues but like the ESG, MIH is synthesized and secreted from these tissues. This experiment also supports the growing body of literature that mTOR inhibition activates downstream transcription factors which are important in maintaining energy homeostasis in times of environmental stress.
Open Access
Analysis of genome-wide targets of Arabidopsis signal responsive 1 (AtSR1) transcription factor and its transcript stability in response to stress
(Colorado State University. Libraries, 2017) Abdel-Hameed, Amira, author; Reddy, A. S. N., advisor; Bush, Daniel, committee member; Leach, Jan, committee member; Abdel-Ghany, Salah, committee member
Abiotic and biotic stresses cause significant yield losses in all crops. Acquisition of stress tolerance in plants requires rapid reprogramming of gene expression. SR1/CAMTA3, a member of signal responsive transcription factors (TFs), functions both as a positive and a negative regulator of biotic stress responses and as a positive regulator of cold stress-induced gene expression. Using high throughput RNA-seq, we identified ~3000 SR1-regulated genes. Promoters of about 60% of the differentially expressed genes have a known DNA binding site for SR1, suggesting that they are likely direct targets. Gene ontology analysis of SR1-regulated genes confirmed previously known functions of SR1 and uncovered a potential role for this TF in salt stress. Our results showed that SR1 mutant is more tolerant to salt stress than the wild type and complemented line. Improved tolerance of sr1 seedlings to salt is accompanied with the induction of salt-responsive genes. Furthermore, ChIP-PCR results showed that SR1 binds to promoters of several salt-responsive genes. These results suggest that SR1 acts as a negative regulator of salt tolerance by directly repressing the expression of salt-responsive genes. Overall, this study identified SR1-regulated genes globally and uncovered a previously uncharacterized role for SR1 in salt stress response. Soil salinity, one of the most prevalent environmental stresses, causes enormous losses in global crop yields every year. Therefore, it is imperative to generate salt tolerant cultivars. To achieve this goal, it is essential to understand the mechanisms by which plants respond to and cope with salt stress. Stress-induced reprogramming of gene expression at multiple levels contributes to the survival of plants under adverse environmental conditions. The control of mRNA stability is one of the post-transcriptional mechanisms that is highly regulated under stress conditions leading to changes in expression pattern of many genes. In this study, we show that salt stress increases the level of SR1 mRNA, by enhancing its stability. Multiple lines of evidence indicate that ROS generated by NADPH oxidase activity mediate salt-induced SR1 transcript stability. Furthermore, cycloheximide (CHX), a protein synthesis inhibitor, also increased SR1 mRNA stability, albeit to a higher level than in the presence of salt, suggesting a role for one or more labile proteins in SR1 mRNA turnover. Similar to salt, ROS generated by NADPH oxidase is also involved in CHX-induced SR1 mRNA accumulation. To gain further insights into mechanisms involved in saltand CHX-induced SR1 stability, the roles of different mRNA degradation pathways were examined in mutants that are impaired in either nonsense-mediated decay (NMD) or mRNA decapping pathways. These studies have revealed that neither the NMD pathway nor the decapping of SR1 mRNA is required for its decay. However, decapping activity is required for saltand CHXaccumulation of SR1 mRNA. To identify any specific regions within the open reading frame of the SR1 transcript (~3 kb) that are responsible for the salt-induced accumulation of SR1 mRNA, we generated transgenic lines expressing several truncated versions of the SR1 coding region in the sr1 mutant background. Then, we analyzed accumulation of each version in response to salt stress and CHX. Interestingly, we identified a 500 nts region in the 3' end of the SR1 coding sequence to be required for both saltand CHX-induced stability of SR1 mRNA. Potential mechanisms by which this region confers SR1 transcript stability in response to salt and CHX are discussed.
Open Access
Analysis of the relationship between genomic instability, heterozygosity levels and phenotype in Saccharomyces cerevisiae
(Colorado State University. Libraries, 2018) Sampaio, Nadia Maria Vieira, author; Argueso, Juan Lucas, advisor; Stargell, Laurie A., committee member; McKay, John K., committee member; Reardon, Kenneth F., committee member
Understanding the forces that mediate genome evolution is a central problem in genetics, with implications for diverse processes that range from speciation, to biotechnological applications, to human disease. The central theme of my dissertation was the characterization of two forces, genomic instability and natural selection, that significantly impact genome structure by influencing the levels of genomic heterozygosity. While genomic instability processes can act to erode heterozygosity from the genome, natural selection may favor the maintenance of heterozygous alleles in cases where there is a positive correlation between heterozygosity and higher fitness. In Chapter I, I reviewed different types of mitotic mutations that can result in the appearance of tracts of homozygosity in genomes and recent discoveries about the temporal accumulation of such events. I also introduce the concept of heterosis, a phenomenon characterized by a positive correlation between genomic heterozygosity and phenotype in many species, and its potential role in contributing to the long-term maintenance of genomic heterozygosity. In Chapter II, I describe the characterization of a mechanism of systemic genomic instability in yeast that challenges the conventional model of gradual and independent accumulation of mutations. We showed that a subset of mitotic cells within a population experience bursts of genomic instability, which results in multiple independent events of loss-of-heterozygosity (LOH) accumulating over one or a few generations of mitotic cell division. We named this outcome "systemic genomic instability". The occurrence of this phenomenon was initially identified in the heterozygous yeast strain JAY270, and then validated in a conventional laboratory strain background, whose genome is almost fully homozygous. Elevated rates of coincident LOH was also observed in mutant strains incapable of entering meiosis, indicating cryptic initiation of meiotic recombination followed by return-to-growth in a few cells in the population was not responsible for the higher than expected rates of coincident LOH. This finding brings to light a novel and intriguing mechanism of genomic instability in yeast that has relevant parallels to bursts of accumulation of copy number alterations in the human genome, providing a powerful experimental model system to dissect the fundamental mechanisms responsible for the generation of rapid changes in chromosome structure. In Chapter III, we explored the role that genomic heterozygosity plays on the superior industrial traits of the JAY270 strain. In the previous Chapter we showed that mitotic recombination leading to LOH occurs at a high frequency during JAY270's clonal propagation. These LOH events act against the long-term maintenance of genomic heterozygosity, yet about 60% of JAY270's genome has remained heterozygous over time. We hypothesized that specific heterozygous alleles may have a positive impact on the traits of this strain and therefore were maintained through selection. We generated a collection of inbred strains derived from JAY270, and assessed them phenotypically under different growth conditions. Our results demonstrated that genomic heterozygosity indeed has a substantial impact on two important industrial traits of this strain – heat stress tolerance and growth kinetics. We identified several genomic regions potentially associated with those traits and conducted experiments to investigate the bulk contributions of heterozygosity blocks in three specific chromosomes. This study revealed candidate regions containing loci that potentially underlie important industrial traits of JAY270 and details on the extent to which heterozygosity may impact JAY270's genome evolution and phenotype. The combined results of these research projects provide important insights about the role of genomic instability mechanisms and their phenotypic outcomes in determining genome evolution, contributing discoveries that may have important practical implications for diverse fields, including biotechnology, cancer development and evolution, as well as genome sciences.
Open Access
Antibacterial growth effects and speciation of several vanadium salts and complexes
(Colorado State University. Libraries, 2023) Arhouma, Zeyad Kamal, author; Crans, Debbie C., advisor; Crick, Dean, committee member; Roess, Deborah, committee member; Jackson, Mary, committee member
Vanadium (V) is a first-row transition metal ion that acts as a phosphatase inhibitor with a wide variety of biochemical and physiological functions. The ability of vanadium to form stable polyoxovanadates (POVs) and organometallic complexes has attracted attention for studying the properties and effects of these compounds in various biological systems. In my research, I used the bacterium species Mycobacterium smegmatis (M. smeg), which has undergone reclassification and is now classified as Mycolicibacterium smegmatis. Despite the taxonomic change, both the previous and current classifications use the same abbreviation, M. smeg. I also carried out some studies in Mycobacterium tuberculosis (M. tb). In my work, I explored the properties of several types of vanadium compounds including salts, oxometalates, and coordination complexes to investigate how they impact cellular growth. The first chapter of this dissertation focuses on determining the growth inhibitory effects of decavanadate (V10) and rapidly exchanging oxovanadates on the growth of two mycobacterial species: M. tb and M. smeg. Speciation analysis, utilizing 51V NMR spectroscopy, was employed to document that one specific oxometalate exhibits greater potency as a growth inhibitor for these mycobacterial species compared to other oxovanadates, indicating selectivity in its cellular interaction. Oxometalates have been involved in numerous applications in biological and medical studies, including their ability in addressing the phase-problem in X-ray crystallography of the ribosome. This study investigated the effect of different vanadate salts on the growth of M. smeg and M. tb, highlighting the critical role of speciation in the observed growth inhibition. Specifically, the large orange-colored sodium decavanadate (V10O286−) anion was found to be a stronger growth inhibitor for these bacteria compared to the colorless oxovanadate derived from sodium metavanadate. The 51V NMR spectroscopy and speciation calculations were employed to monitor the vanadium(V) speciation in the growth media and its conversion among species under growth conditions. Our results show that the decavanadate was 200-20 times more potent in inhibiting growth dependent the consideration of molecules or total vanadium content. The findings presented in this work are particularly important in the context of the numerous applications of polyoxometalates in biological and medical studies. The second chapter focuses on investigating the inhibitory effects of two monosubstituted decavanadates (V10): monoplatino(IV)nonavanadate(V) ([H2PtIVV9O28]5−, V9Pt), and by MoIV in monomolybdo(VI)nonavanadate(V) ([MoVIV9O28]5−,V9Mo) on the growth of M. smeg. The inhibitory effects of V9Pt and V9Mo were examined against the growth of M. smeg with EC50 values of 0.0048 mM and 0.015 mM, respectively. These values were compared to the reported inhibitory value of decavanadate ([V10O28]6−/[HV10O28]5−, V10) on M. smeg (EC50 = 0.0037 mM). Time-dependent 51V NMR spectroscopic studies were carried out for all three polyanions in aqueous solution, biological medium (7H9), and heated and non-heated supernatant. These studies aimed to evaluate their stability in their respective media, monitor their hydrolysis over time to form different oxovanadates, and calculate the corresponding EC50 values. The results presented in this study indicate that the two related derived decavanadate derivatives (V9Pt and V9Mo) and V10 exhibited greater potency as growth inhibitors of M. smeg, compared to monomeric vanadate (V1). The spectroscopic characterization conducted in the growth medium led to the conclusion that both the decavanadate structure and its properties play significant roles in their growth effects. In the third chapter in this dissertation, we investigated the growth effects of an anticarcinogenic non-toxic Schiff base oxidovanadium(V) complex (N-(salicylideneaminato)-N'-(2-hydroxyethyl) ethane-1,2-diamine) coordinated to the 3,5-di-tert-butylcatecholato ligand on a representative bacterium, M. smeg.. In addition, we synthesized a series of the Schiff base V-complexes based on previously reported methods and examined the effect of complexes as well as the free catecholates on the bacterial growth. To determine the inhibition activity of these complexes on M. smeg., the biological studies were complemented by spectroscopic studies using UV-Vis spectrophotometry and NMR spectroscopy. These spectroscopic studies determine which complexes remained intact under biologically relevant conditions. In this work, we examine (1) the growth effects of Schiff base oxidovanadium complexes coordinated to a catechol, (2) the growth effects of the respective free catecholates on M. smeg., and (3) the effects of the scaffold. These studies allowed us to demonstrate that some metal coordination complex exhibited greater potency than the ligand alone under biological conditions, whereas others showed greater effects of the free catecholate ligand and in one case the effects were similar of complex and catecholate ligand. The findings from these studies revealed that the observed effects of the Schiff base V-catecholate complex were influenced by the properties of the catechol, including toxicity, hydrophobicity, and steric factors. Finally, the fourth chapter presents preliminary research data on the antimicrobial effects of two pseudospherical mixed-valence polyoxovanadates (MV-POVs), namely K(NH4)4[H6PVIV2VV12O42]·11H2O (V14) and (Me4N)6[VIV8VV7O36(Cl)] (V15) on the growth of M. smeg. These MV-POVs showed complex effects on cell growth, as many of these systems are not stable under biological conditions. To investigate the vanadium(V) speciation in aqueous solutions and growth media, as well as to monitor any conversion among species under growth conditions, 51V NMR spectroscopy was employed. The 51V NMR spectra revealed some hydrolysis and more extensive oxidation of vanadium(IV) in V14 compared to V15 in both aqueous solutions and media. The studies show that both MV-POVs are effective growth inhibitors. The combined findings from the studies described in all the chapters of this dissertation indicate that the stability of the vanadium compound and its structure plays a significant role in the ability of the vanadium complexes to inhibit bacterial growth. These studies highlight the importance of speciation in the biological activity of vanadium complexes.
Open Access
Applications of inorganic nanoparticles in diabetes
(Colorado State University. Libraries, 2016) Elhabush, Nada Atiya Omar, author; Crans, Debbie C., advisor; Barisas, George B., committee member; Roess, Deborah A., committee member
Diabetes Mellitus (DM) is an endocrine and metabolic disease that has become a global emergency because of the rapid rise in morbidity and mortality rates worldwide. Since the direct delivery of biomolecules, such as insulin, to treat DM is inefficient and subjected to enzymatic degradation, nanotechnology and nanomedicine research have been devoted to the development of more effective methods to treat DM. Nanoparticles (NP), organic, inorganic, or hybrid, have served as potential carrier for safe and efficient transport for insulin. Additionally, several NP have biological activities that help treat and/or prevent DM and diabetes complications, such as antioxidant, anti-apoptotic, or insulin-mimetic activities. Moreover, physicochemical properties of some NP allow them to be used in diagnostic tools for potential diagnosis or monitoring purposes. This work highlights the applications of inorganic NP such as, gold, selenium, silver, calcium phosphate, zinc oxide, cerium oxide, and iron oxide and in the treatment or diagnosis of DM.
Open Access
Assessing and understanding the generation and function of RNA decay intermediates in non-insect borne flaviviruses
(Colorado State University. Libraries, 2019) Mundell, Cary T., author; Wilusz, Jeffrey, advisor; Geiss, Brian, committee member; Perera, Rushika, committee member; Reddy, Anireddy, committee member
Cellular gene expression is an intricate process regulated on many levels that allows the cell to react correctly to stimuli or to maintain homeostasis. RNA viruses must act to preferentially drive production of their own messenger RNAs (mRNAs) and proteins in order to successfully replicate and ensure continued infection. Due to the necessity for RNA viruses to remain in the cytoplasm, regulatory factors that affect host mRNAs likely also affect the transcripts of RNA viruses. RNA decay represents a major pathway of regulation for mRNAs. A multitude of RNA viruses possess unique mechanisms that act to prevent the decay of viral transcripts and allow for successful translation. Members of the viral family Flaviviridae are positive sense, single-stranded RNA viruses that do not possess a poly(A) tail. Therefore, it is highly likely that these transcripts would be marked as deadenylated and shuttled down one of the RNA decay pathways that exist in the cell. Interestingly, members of the genera Flavivirus of the family Flaviviridae possess a conserved structured 3' untranslated region (UTR) that acts to interfere with the decay processes of the major cytoplasmic cellular 5'-3' decay enzyme XRN1. In addition, members of the generas Hepacivirus, Hepatitis C Virus (HCV) and Pestivirus, Bovine Viral Diarrhea Virus (BVDV), possess XRN1 stalling elements within their 5' UTRs. These stalling sites block the action of the exonuclease and generate decay intermediates. The generation of these decay intermediates represses XRN1 activity in the infected cell. Herein we demonstrate a new method for studying RNA decay through the use of XRN1-resistant RNAs (xrRNAs). In this method we utilize the well characterized xrRNA of Dengue Virus Type 2 (DENV2) as a readout to study the decay rates of relatively large RNA constructs. We show that not only is utilizing an xrRNA an effective method for confirming XRN1-mediated decay, but that the accumulation of the readout xrRNA can be utilized to understand changes in the decay kinetics of RNA substrates. We further utilize this method to demonstrate a lack of XRN1 stalling elements within the poliovirus internal ribosomal entry site (IRES) element. We provide evidence that the stalling of XRN1 in the 5' UTR of BVDV is dependent on both the presence of the entire IRES structure and the presence of a stem loop 5' to the IRES element through the analysis of a series of truncations. Finally, we demonstrate one possible role for the HCV and BVDV decay intermediates as the truncated IRES element maintains translatability in an in vitro system. Collectively, these data better define the structural requirements for the novel XRN1 stalling elements located in the 5' UTR of non-insect borne members of the Flaviviridae as well as the potential function of the decay intermediates.
Open Access
Autism-associated δ-catenin G34S mutation promotes GSK3β-mediated premature δ-catenin degradation inducing neuronal dysfunction
(Colorado State University. Libraries, 2019) Nip, Kaila, author; Kim, Seonil, advisor; Bamburg, James, committee member; Tsunoda, Susan, committee member
δ-catenin is a crucial component of a synaptic scaffolding complex, which regulates synaptic structure and function in neurons. Loss of δ-catenin function is strongly associated with severe autism spectrum disorder (ASD) in female-enriched multiple families. In particular, a G34S (Glycine 34 to Serine) mutation in the δ-catenin gene has been identified in ASD patients and suggested to exhibit loss-of-function. The G34S mutation is located in the amino terminal region of δ-catenin, where there are no known protein interaction domains and post-translational modifications. Notably, the Group-based Prediction System predicts that the G34S mutation is an additional target for GSK3β-mediated phosphorylation, which may result in protein degradation. Therefore, we hypothesize the G34S mutation accelerates δ-catenin degradation, resulting in loss of δ-catenin function in ASD. Indeed, we found significantly lower G34S δ-catenin levels compared to wild-type (WT) δ-catenin when expressed in cells lacking endogenous δ-catenin, which is rescued by genetic inhibition of GSK3β. By using Ca2+ imaging in cultured mouse hippocampal neurons, we further revealed overexpression of WT δ-catenin is able to significantly increase neuronal Ca2+ activity. Conversely, Ca2+ activity remains unaffected in G34S δ-catenin overexpression, which is reversed by pharmacological inhibition of GSK3β using lithium. This suggests the G34S mutation of δ-catenin provides an additional GSK3β-mediated phosphorylation site, which could promote δ-catenin premature degradation, resulting in loss-of-function effects on neuronal Ca2+ activity in ASD. In addition, inhibition of GSK3β activity is able to reverse G34S-induced loss of δ-catenin function. Thus, inhibition of GSK3β may be a potential therapeutic treatment for δ-catenin-associated ASD patients.
Open Access
Autophagy modulation: role in anti-cancer therapy
(Colorado State University. Libraries, 2015) Barnard, Rebecca A., author; Gustafson, Daniel L., advisor; Thamm, Douglas H., committee member; Thorburn, Andrew, committee member; Yao, TingTing, committee member
Autophagy is a conserved lysosomal degradation process characterized by cellular self-digestion. Autophagy results in turnover of the cytoplasm allowing for metabolic maintenance and organelle quality control, particularly during cell stress. These aspects of autophagy can facilitate tumor cell survival and resistance. As such, autophagy inhibition is being explored in clinical trials as a novel approach to chemosensitization. However, there are still a number of unresolved concerns in regards to the use of autophagy inhibition as a therapy. It is still unclear how autophagy functions in metastasis development. Therefore, we investigated the role of autophagy in metastasis by modulating autophagy in different mouse models and cell based assays that reflect the steps of metastatic development. We found that autophagy was not required for tumor cell colonization within the site of metastasis nor did autophagy alter the metastatic capabilities of the cells. Rather, autophagy appeared to impact the pre-metastatic environment through effects on bone marrow derived cell number which mediate the establishment of the metastatic niche. Stimulating autophagy, before tumor cells disseminated, could speed metastatic development and increase the number of these cells within circulation and eventual sites of metastasis. Correspondingly, inhibiting autophagy could delay metastasis and reduce circulating bone marrow derived cells. These studies suggest that autophagy is most critical in the stages prior to tumor cell arrival at the site of metastasis, by influencing the metastatic microenvironment. While increased autophagy is often considered to be a common tumor adaptation, it is now apparent that some tumor types are more dependent on autophagy than others. However, it is not well understood which tumors these are. Triple negative, Stat3 activated breast cancers were identified as autophagy dependent by collaborator Dr. Paola Maycotte. We tested the efficacy of autophagy inhibitor chloroquine (CQ) in xenograft models of triple negative and estrogen receptor positive breast cancer. CQ was only efficacious in the triple negative tumors. As some canine osteosarcomas also have constitutive Stat3 activity, we assessed the relationship of Stat3 activity and CQ sensitivity. Unlike in breast cancer, Stat3 phosphorylation did not indicate increased sensitivity to CQ in canine osteosarcoma. However, all the osteosarcoma cell lines responded to treatment. Using microarray analysis we identified potential compensatory pathways that have been previously reported to work in concert with autophagy in other cell types and may serve as useful combinational therapies. Currently, the only autophagy inhibitor available clinically is CQ or derivative hydroxycholorquine (HCQ). It is still uncertain whether these drugs can actually achieve autophagy inhibition in patients. Dogs serve as a good model for human cancer and there is an unmet need for novel therapies in the treatment of canine lymphoma. Thus we conducted a phase I clinical trial in canine lymphoma patients with the goals of finding a maximum tolerated dose in combination with doxorubicin (DOX) and the relationship of HCQ concentration and autophagy inhibition. We found that this combination can be well tolerated with a 20% reduction in DOX. HCQ can achieve autophagy inhibition in patients, but not consistently. There appears to be a threshold requirement of HCQ needed in order to effectively inhibit autophagy. There was a suggestion of efficacy as response rate was superior to historical data employing DOX alone. Therefore autophagy inhibition warrants further clinical study as an anti-cancer therapy.
Open Access
Biology, comparative genomics and molecular diagnostics of Xanthomonas species infecting rice and corn
(Colorado State University. Libraries, 2017) Lang, Jillian M., author; Leach, Jan E., advisor; Bush, Daniel, committee member; Reddy, Anireddy, committee member; Verdier, Valérie, committee member
Emerging bacterial diseases on staple and economically important crops can pose critical threats to food security. Accurate identification of bacterial plant pathogens is the foundation of effective management for growers. This work advances the application of genomics to identify and characterize bacterial plant pathogens in the genus Xanthomonas that can cause destructive diseases on most agricultural crops, including rice and corn. In this thesis, taxonomy, host range, disease phenotypes and basic biology of the following pathogens were established: X. oryzae pv. oryzae, X. o. pv. oryzicola, X. o. pv. leersiae and X. vasicola pv. vasculorum. X. o. pv. oryzae and X. o. pv. oryzicola infect rice and cause bacterial blight and bacterial leaf streak, respectively. X. o. pv. leersiae infects cutgrass (Leersia sp.), weedy grasses that can serve as alternative hosts to X. oryzae and are endemic in all rice growing regions. X. vasicola pv. vasculorum was identified as the causal agent of bacterial leaf streak of corn, an emerging and now wide-spread disease in the United States, that was reported for the first time in 2017. This work established that X. vasicola pv. vasculorum can also infect sorghum and sugarcane and that the US strain is 99% similar to strains isolated over 20 years ago in S. Africa. To develop robust molecular diagnostic tools for these pathogens, unique features needed to be first identified. Using comparative genomics that included closely related bacteria and distant relatives, PCR-based diagnostic tools were developed, then validated using isolated cultures and field grown plant materials. Comparative genomics also contributed to elucidation of the taxonomy and phylogeny of X. o. pv. leersiae and X. v. pv. vasculorum. Characterization of X. o. pv. leersiae revealed adaptations to both the weedy grass hosts and rice. These features include virulence proteins that target homologous host genes (transcription activator like effectors, TALEs) to influence host gene expression. I conclude that X. oryzae is a complex that includes X. oryzae pv. oryzae, X. o. pv. oryzicola and X. o. pv. leersiae and that this complex can provide a unique window into pathogen evolution. By better understanding how pathogens adapt to their environments including new hosts, growers can manage surrounding ecosystems more effectively to minimize yield losses and therefore, contribute to food security.
Open Access
Biophysical studies of motions and interactions of membrane proteins
(Colorado State University. Libraries, 2011) Winter, Peter William, author; Barisas, B. George, advisor; Roess, Deborah A., advisor; Crans, Debbie C., committee member; Bamburg, James R., committee member
We have utilized a variety of biophysical techniques to quantitatively examine the motions and interactions of transmembrane proteins on living cells at the single-molecule level. These include both widefield and confocal optical microscopic methods such as single particle tracking, Förster resonance energy transfer and ratiometric imaging of phase-sensitive probes of lipid order, together with spectroscopic fluctuation methods such as fluorescence correlation spectroscopy and photon counting histogram analysis. Our studies indicate that; 1. Luteinizing hormone receptors on CHO cells and KGN human granulosa cells exhibit restricted lateral diffusion and increased self-association after exposure to human chorianic gonadotropin; 2. In addition to insulin receptor and IGF1 receptor homodimers, rat basophilic leukemia 2H3 cells express significant levels of insulin receptor-IGF1 receptor heterodimers; 3. Clustering of insulin receptors after exposure to insulin on rat basophilic leukemia 2H3 cells is affected by disruption of actin-filaments but not by extraction of membrane cholesterol; 4. Chromium Picolinate and Bis(maltolato)oxovandium(IV) both restrict the lateral diffusion of insulin receptors on rat basophilic leukemia cells and; 5. Individual FcE receptors on rat basophilic leukemia cells exhibit orientation fluctuations on millisecond timescales.
Embargo
Characterization and insights into the molecular mechanism of cytokinin-induced priming of plant defenses
(Colorado State University. Libraries, 2023) McIntyre, Kathryn, author; Argueso, Cristiana, advisor; Bush, Daniel, committee member; Leach, Jan, committee member; Stewart, Jane, committee member; Reddy, Anireddy, committee member
Plants have developed several mechanisms to cope with pathogenic challenges. One of these mechanisms, known as defense priming can be effective at reducing susceptibility to pathogens. Compared to unprimed plants, the immune response from primed plants, upon pathogen attack, is much stronger. This mechanism of induced disease resistance can be initiated by biological and chemical agents. The major benefit of priming is the induction of a high level of protection with considerably low fitness costs making it an attractive disease management strategy to preserve agricultural output. Recent research has demonstrated that the plant hormone cytokinin (CK) has a priming effect against biotrophic pathogens, a phenomenon referred to here as cytokinin-induced priming (CIP). This dissertation aims to gain further understanding of CIP against the hemibiotrophic bacterial pathogens Pseudomonas syringae pv. tomato (Pst) and Pseudomonas syringae pv. maculicola (Psm) in Arabidopsis thaliana (Arabidopsis) and Brassica napus, respectively as well as the necrotrophic fungal pathogen Botrytis cinerea in Arabidopsis. Chapter 2 focuses on characterizing CIP as a true priming agent by investigating the timeframe in which CIP is most effective at reducing susceptibility to Pst and Psm in both Arabidopsis and its closely related relative, B. napus and the impacts on plant growth due to CIP in these pathosystems. Moreover, we discovered that other known priming agents depend on endogenous CK signaling suggesting CK-mediated processes are involved in the priming of defense responses. The role of CK in primed defenses against B. cinerea is explored in chapter 3 where CIP is demonstrated to reduce necrotic lesion size caused by B. cinerea in a manner dependent on the JA-mediated defenses and partially on SA-mediated defenses. Transcriptome analysis revealed that during the priming stage, CK prepares the plants for pathogenic challenge through the accumulation of cellular components needed for translation and metabolites utilized for energy production and defense. Following B. cinerea inoculation, CIP suppresses defense while increasing photosynthetic-related processes. In the final chapter, molecular mechanisms are explored during CIP against Pst. Through transcriptome changes, priming by CK potentiates gene expression associated with systemic induction of defense, also known as systemic acquired resistance (SAR), following Pst challenge. Using this information, it is demonstrated that CK treatment can also induce SAR and that the known SAR inducer, L-pipecolic acid, is dependent on endogenous CK signaling. Due to the previously identified relationship between CK and source-sink relationships, amino acid transport was demonstrated to have a role in both CIP and CK-induced SAR. New agricultural practices that mitigate crop loss due to plant diseases are beneficial in terms of sustainability and economic costs. The use of CK as a priming agent offers an avenue for a new disease management strategy in that CIP protects plants against a broad range of pathogens with minimal effects on plant growth. The molecular mechanisms underlying CIP discovered here offers new insights into the relationship between plant metabolism and defense, where its exploitation could be used to create disease protection strategies.
Open Access
Characterization of biased partner choice in mitotic non-allelic homologous recombination of Saccharomyces cerevisiae
(Colorado State University. Libraries, 2023) Merriman, Sean, author; Argueso, Juan Lucas, advisor; Markus, Steven, committee member; Nishimura, Erin, committee member; Wiese, Claudia, committee member
Using yeast as a model in which to study copy number variation (CNV)-generating mutations, the J.L. Argueso lab has discovered that a specific region of S. cerevisiae genome (the right arm of chromosome 7; Chr7R) is much more susceptible to sustaining deletions as a translocation recipient than other apparently similar segments of the genome. Further, Chr7R acquires amplifications as a translocation donor less frequently than other chromosomes. To begin unraveling the cause of this unusual behavior, we evaluated the effect of several candidate genes involved in chromatin mobility and sister chromatid cohesion on the mutational spectra involving Chr7R. Our results suggest that regulatory factors of chromatin mobility or sister chromatid cohesion affect the outcomes of HR-mediated repair events at Ch7R. We are hopeful that our findings will open a window into the fundamental cellular processes that are responsible for CNVs found in eukaryotic genomes, and inform translational implications for modeling this class of mutation in cancer.
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.
Embargo
Characterization of modes and kinetics of mutation accumulation in Saccharomyces cerevisiae through the analysis of defined cellular lineages
(Colorado State University. Libraries, 2024) Stewart, Joseph, author; Argueso, Juan Lucas, advisor; Moreno, Julie, committee member; Regan, Daniel, committee member; Wiese, Claudia, committee member
In the field of evolution, gradualism is the process of incremental adaptation supported by a slow and random accumulation of mutations that, over time, lead to genetic diversification and fitness gains. Although this Darwinian model is well supported and widely accepted, it cannot always explain the rapid changes seen in some instances such as tumors with extremely high and complex mutation loads. Recent reports in various organisms, including from our group using Saccharomyces cerevisiae, provide evidence for an additional mode of rapid and non-independent accumulation of chromosomal rearrangements. We have used a yeast model to follow the accumulation of structural genomic rearrangements such as loss of heterozygosity (LOH). We found that while chances of a single LOH event happening are very low, two or more LOH tracts co-occurred at rates 25- to 200-fold higher than expected if these events were independent of each other; therefore, the conventional process of slow and independent accumulation of mutations are not sufficient to account for every change in the genome. In the present study, we focused on temporal kinetics of bursts of LOH accumulation in yeast. We developed a hybrid diploid yeast experimental strain that enables identification of LOH event both through counter-selection and visual screening for colony color. This hybrid strain, made from the S288c and SK1 genetic backgrounds, possesses ~55,000 heterozygous SNPs distributed throughout the genome and allows for ease of tracking LOH events through sequencing. The screening approach was used in combination with microcultures (one cell grown for 5 or 6 divisions) in phylogenetic analyses that unambiguously revealed 18 cases where multiple LOH events co-occurred in the same cell division cycle. Collectively, these studies offer support for punctuated bursts of mutation accumulation caused by systemic genomic instability (SGI). Additionally, we have investigated a potential mechanism that influences SGI, namely global noise in gene expression.
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.
Open Access
Characterization of zebrafish models of filamin C related cardiomyopathy
(Colorado State University. Libraries, 2019) Alnefaie, Rasha M., author; Garrity, Deborah, advisor; Reddy, A. S. N., committee member; Lybourn, Paul, committee member; Muller, Racheal, committee member
Cardiomyopathies are a group of cardiac muscle diseases characterized by abnormal function and/or structure of the myocardium which cause arrhythmia, heart failure or sudden death. In many cases, cardiomyopathy is a genetic disease and the majority of inherited cases are caused by mutations in genes that encode cardiac costameric and sarcomeric proteins. Cardiomyopathies include different types, such as dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM), and arrythmogenic cardiomyopathy (ARCM). These groups of disorders can have common cellular phenotypes and mechanisms. To date, few studies have described the roles of filamin C in cardiac development or explained how mutations in filamin C cause cardiomyopathic disease. Due to the lack of a suitable animal model, the pathological mechanisms underlying this disease and the role of filamin C in cardiac development remain unclear. Here, we created a zebrafish loss-of-function model for two flnc paralogous genes. We investigated several genetic lines bearing mutations in filamin C that target different sites of the gene. As for humans, zebrafish mutants exhibited variable penetrance and variable expressivity. Double flnca and flncb mutant hearts exhibited more pronounced cardiac morphological defects compared to single mutants, leading to the conclusion that these paralogs play redundant roles in zebrafish heart development. The cardiac morphological phenotype of double flnc mutant embryos is characterized by a decrease in cardiac output and stroke volume as is also observed in patients who suffer from cardiomyopathies. Using a transgenic line expressing GFP in cardiac z-discs, we find that knockdown of flnca and flncb via morpholinos and double flnca and flncb mutant hearts exhibited irregular z-discs. In support of this finding, ultrastructural analysis by transmission electronic microscopy for flncb morphant embryos and double flnca and flncb mutants indicated disorganized myofibrils with fewer consecutive sarcomeres. Particularly, z-discs were irregular or apparently absent, and numerous small vacuoles and potentially autophagous vesicles were observed. Additionally, through double flnca and flncb mutant, we demonstrated that filamin C is required for normal cardiomyocyte morphology and microfilament arrangement. In summary, the zebrafish model demonstrates an essential requirement for filamin C function during heart and skeletal muscle development. Depletion of filamin C impairs both sarcomerogenesis and alters the cytoskeletal architecture of cardiomyocytes.