Cellular & Molecular Biology Doctoral Work

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    Glutathione S-Transferase Pi-1 in Pancreatic Cancer Pathogenesis: Mechanistic Insights Into Its Role in Oxidative Stress, Cell Signaling, and Metabolic Pathways
    (North Dakota State University, 2021) Singh, Rahul
    Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related deaths in the US. Although surgery remains the most effective treatment option, only a few PDACs are resectable at diagnosis. Further, only a few PDAC patients respond to conventional chemotherapy making long-term survival notably challenging. Therefore, the identification of novel therapeutic targets is needed to improve PDAC treatment efficacy. Here, we investigated the role of glutathione S-transferase pi-1 (GSTP1) in PDAC pathogenicity. We postulated that a higher expression of GSTP1 provides selective advantages to PDAC cells by scavenging excessive reactive oxygen species (ROS) and promoting survival. Using shRNAs, we knocked down the expression of GSTP1 in metabolically diverse PDAC cells. We show loss of GSTP1 reduces PDAC cell growth and causes oxidative stress. Our results provide evidence that GSTP1 knockdown activates apoptotic signaling by phosphorylating c-Jun N-terminal kinase (JNK) and c-Jun. Further, supporting in vitro data, nude mice bearing orthotopically implanted GSTP1 knockdown PDAC cells showed a significant reduction in tumor size and volume and reduced Ki67 staining. Further, using multi-omic techniques, we show that GSTP1 knockdown significantly changes the global transcriptomic and proteomic signatures of PDAC cells. Gene set enrichment analyses revealed that cellular metabolism and energy production pathways are most affected in GSTP1 knockdown PDAC cells. Specifically, we report a reduction in the mRNA and protein expression of aldehyde dehydrogenase 7A1 (ALDH7A1) and solute carrier 2A3 (SLC2A3) in GSTP1 knockdown cells compared to the control. We propose that the growth-inhibitory effects of GSTP1 knockdown are due to redox imbalance and impaired energy production pathways. Our data are the first steps of validating GSTP1 as a potential therapeutic target for PDAC. Collectively, our results provide evidence that GSTP1 inhibitors combined with conventional chemotherapy can be an effective treatment for PDAC patients. However, understanding the function of GSTP1 in cancer cell metabolism requires further investigations. Because GSTP1 knockdown affects various metabolic genes, we will evaluate the bioenergetic changes in GSTP1 knockdown PDAC cells. Additionally, comparative evaluation of metabolites and lipids in control and GSTP1 knockdown cells will expand our knowledge of GSTP1 in cancer cell physiology and metabolism.
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    Biochemical and Cellular Characterization of Replication Factor A (RFA) During Meiosis and The DNA Damage Response in Saccharomyces cerevisiae
    (North Dakota State University, 2021) Adsero, Angela Marie
    Replication Factor A (RFA) is an essential heterotrimeric single-stranded DNA (ssDNA) binding complex, comprised of Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae. RFA is required for DNA replication, repair, recombination, and cell cycle regulation. RFA acts as a sensor of ssDNA, a common intermediate of these processes, and coordinates these processes through recruitment of proteins. For example, during the DNA damage response (DDR), RFA-coated ssDNA is necessary for the recruitment and activation of the sensor kinase Mec1. Additional checkpoint proteins, also recruited by RFA, are necessary for the downstream recruitment and activation of the effector kinase Rad53 that ultimately leads to cell cycle arrest. Thus, RFA acts as a bridge to recruit the proteins required for checkpoint regulation in response to DNA damage. Importantly, cell cycle resumption is contingent on Rad53 deactivation. There are two known scenarios in which Rad53 is deactivated: (1) checkpoint recovery, in which cells resume the cell cycle after DNA repair or (2) checkpoint adaptation, in which cells proceed with the cell cycle despite the continued presence of irreparable DNA damage. Previous work has demonstrated that cells undergoing checkpoint adaptation display late Rfa2 N-terminal (NT) phosphorylation that is correlated with the inactivation (dephosphorylation) of Rad53. Additionally, the use of rfa2 NT mutations consistently demonstrate that a negatively charged NT promotes adaptation in all adaptation-deficient strain backgrounds investigated. Interestingly, Rfa2 NT phosphorylation also occurs early during meiosis. This work demonstrates that: (1) Rfa1-DBD-F participates in protein-protein interactions that are sensitive to DNA damage, (2) Rfa2 phosphorylation increases the DNA damage sensitivity of mutants with deficient DNA damage checkpoints, (3) the Rfa2 NT is required for proper progression through meiosis that appears to be unrelated to RFA functions in replication or DNA repair by homologous recombination (HR), and (4) Rfa2 phosphorylation may regulate Mec1 checkpoint signaling during the DDR to control checkpoint exit and cell cycle resumption. A mechanism is proposed that considers both Rfa1 DBD-F and the Rfa2 NT involvement to initiate HR repair that essentially allows for the continuation of the cell cycle by the delocalization of Mec1.
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    Changing the Pancreatic Cancer Treatment Paradigm: Developing Clostridium novyi as an Intravenously Injectable Solid-State Tumor Therapeutic
    (North Dakota State University, 2020) Dailey, Kaitlin Marie
    The development of a drug able to distinguish between tumor and host cells has been long sought, but the solid tumor microenvironment (TME) confounds many current therapeutics. Solid tumors present several challenges for oncotherapeutics, primarily, (1) aberrant vascularization, resulting in hypoxia, necrosis, abnormally high pH, and (2) tumor immune suppression. Oncolytic microbes are drawn to this microenvironment by an innate ability to selectively penetrate, colonize, and eradicate solid tumors as well as reactivate tumor associated immune components. To consider oncolytic bacteria deployment into this microenvironment, Chapter 1 dives into the background of oncolytic microbes. A discussion of the oncolytic bacterial field state, identifying Clostridium novyi¸ as a promising species, and details genetic engineering techniques to develop customized bacteria. Despite the promise of C.novyi in preclinical/clinical trials when administered intratumorally, the genetic and biochemical uniqueness of C.novyi necessitated the development of new methodologies to facilitate more widespread acceptance. Chapter 2 reports the development of methods that facilitate experimental work and therapeutic translation of C.novyi, including the ability to work with this obligate micro-anaerobe aerobically on the benchtop. While methods development is a necessary step in the clinical translation of C.novyi so too is choosing the correct model of the TME within which to test a potential anti-cancer therapy. While the typical solid TME includes both phenotypic and genotypic heterogeneity, the methods used to model this disease state often do not reflect this complexity. This simplistic approach may have contributed to stagnant five-year survival rates over the past four decades. Nevertheless, simplistic models are a necessary first step in clinical translation. Chapter 3 explores the impact of cancer cell lines co-cultured with C. novyi to establish the efficacy of this oncolytic bacteria in a monolayer culture. Chapter 4 extends this analysis adding not only a level of complexity by using an in vivo model, but also using CRISPR/Cas9 to modify the genome of C.novyi to encode a tumor targeting peptide, RGD, for expression within the spore coat. The combination of these studies indicates that C. novyi is uniquely poised to accomplish the long sought after selective tumor localization via intravenous delivery.
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    Factors Affecting Metabolism During Non-Feeding Stages in Insects
    (North Dakota State University, 2020) Cambron, Liz Doralyn
    Although feeding is important for optimal development and growth in insects, there are several points during the insect life cycle that are non-feeding: metamorphosis, pupation, and overwintering. Non-feeding periods also occur in response to internal cues, such as feedback from nutrient thresholds and immune responses being activated. Additionally, as an insect goes through different developmental stages, its nutritional requirements change in response to or in preparation for non-feeding periods. Most physiological responses like feeding are regulated through an interconnection of pathways, but how these networks change in response to different energy demands, such as immune challenges or changes in metabolism, is poorly understood. One significant pathway that is involved in regulating several physiological processes is the insulin signaling pathway. In my dissertation research, I tested hypotheses explaining the regulation of physiological processes during non-feeding periods in two agriculturally relevant insects, Manduca sexta and Megachile rotundata. First, I investigated how internal cues such as dietary lipid content and immune challenges cause non-feeding periods in M. sexta. Then, I investigated how insulin signaling regulates development during a non-feeding period like overwintering changes in M. rotundata. Since the insulin signaling (IIS) pathway is critical for development and growth, I focused on testing if this pathway plays a role in regulating non-feeding periods. My research showed that increased dietary lipid content causes a cessation of feeding, which suggests there is a possible lipid threshold that when reached, causes M. sexta to switch from lipid consumption for storage to lipid excretion. When looking at another cue like immune challenges, my results showed that during a bacterial infection, a Toll-mediated suppression of IIS pathway may be regulating feeding and causing a non-feeding period exhibited as sickness-induced anorexia. Lastly, my results also showed that the IIS pathway is suppressed in overwintering M. rotundata, and that this process can change in response to temperature. Overall, my dissertation research showed that the insulin signaling pathway and nutrient content play a vital role in regulating non-feeding periods. Investigating insulin signaling, lipid metabolism, and innate immunity in these species closes a gap in knowledge of invertebrate development.
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    The Biology of the Receptor for Advanced Glycation End Products (RAGE) in Cancer
    (North Dakota State University, 2020) Kadasah, Sultan Ftayes Saeed
    Overexpression of the Receptor for Advanced Glycation End Products (RAGE) has been implicated in multiple diseases, including several types of cancer. In different types of cancer, RAGE has been shown to promote cell survival by either autophagy or activation of the transcription factor NF-κB. Based on what is known about RAGE, we hypothesized that the RAGE/ligand interaction at the cell surface promotes pancreatic cancer and melanoma cell survival by both pathways, autophagy and NF-κB activation. To study the role of RAGE in pancreatic cancer resistance to chemotherapy, BxPC-3, MIA PaCa-2, PANC-1, and RAGE overexpressing PANC-1 FLR2 cell-lines were used. A significant decrease in cell viability was observed upon gemcitabine treatment with further significant reduction in cell viability upon combination of gemcitabine with the RAGE inhibitor IgG 2A11. In our studies we showed that RAGE plays a central role in pancreatic cancer cell resistance to gemcitabine by increasing autophagy. To test the importance of RAGE localization in mediating drug resistance, three melanoma cell-lines (WM115, WM266, and SK-MEL2) with their daughters, RAGE overexpressing cells (WM115-RAGE, WM266-RAGE, and SK-MEL2-RAGE) were used. Wild type cell-lines only expressed RAGE intracellularly while RAGE overexpressing cells expressed RAGE both at the cell surface and inside cells. We show in this study that only the cell surface RAGE is involved in melanoma resistance to dacarbazine. We next tested the effects of RAGE/RAGE ligand interaction at the cell surface in pancreatic tumor growth. We used two carcinoma cell-lines, PANC-1 and MIA PaCa-2, for this purpose. Both cell-lines were transiently transfected with a NF-κB/Luciferase reporter plasmid to test the effects of the interaction between RAGE and its ligands on the activation of the NF-κB signaling pathway. We observed higher NF-κB activity upon treatment with RAGE ligands (AGE, S100P, and S100A8/A9) compared to non-treated cells. Higher activity of NF-κB was coupled with a higher expression of cyclin D1 and lower expression of p53, NF-κB target genes.
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    Therapeutic Potential of Piperlongumine for Pancreatic Ductal Adenocarcinoma
    (North Dakota State University, 2019) Mohammad, Jiyan Mageed
    Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies because it is often diagnosed at a late disease stage and has a poor response rate to currently available treatments. Therefore, it is critical to develop new therapeutic approaches that will enhance the efficacy and reduce the toxicity of currently used therapies. Here we aimed to evaluate the therapeutic potential and mechanisms of action for piperlongumine (PL), an alkaloid from long pepper, in PDAC models. We postulated that PL causes PDAC cell death through oxidative stress and complements the therapeutic efficacy of chemotherapeutic agents in PDAC cells. First, we determined that PL is one of the most abundant alkaloids with antitumor properties in the long pepper plant. We also showed PL in combination with gemcitabine, a chemotherapy agent used to treat advanced pancreatic cancer, reduced tumor weight and volume compared to vehicle-control and individual treatments. Further, biochemical analysis, including RNA sequencing and immunohistochemistry, suggested that the antitumor activity of PL was associated with decreased cell proliferation, induction of cell cycle arrest, and oxidative stress-induced cell death. Moreover, we identified that c-Jun N-terminal kinase (JNK) inhibition blocks PL-induced cell death, translocation of Nrf2, and transcriptional activation of HMOX1 in PDAC. Finally, high-throughput drug and CRISPR screenings identified potential targets that could be used in combination with PL to treat PDAC cells. Collectively, our data suggests that cell cycle regulators in combination with PL might be an effective approach to combat pancreatic cancer.
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    Structural Studies of BECN1, A Key Autophagy Protein, and Intrinsically Disordered Regions in Autophagy Proteins
    (North Dakota State University, 2016) Mei, Yang
    Autophagy, a conserved catabolic process required for cellular homeostasis in eukaryotes, is regulated by many proteins. The central goal of my doctoral research is to investigate conformational flexibility of autophagy proteins, with a special focus on BECN1, a core component of the class III phosphatidylinositol-3 kinase autophagosome nucleation complex that may serve as an autophagy interaction hub. Our rigorous bioinformatics analysis predicts that 57% of 59 key human autophagy proteins contain intrinsically disordered regions (IDRs), which lack stable secondary and tertiary structure. The prevalence of IDRs suggests that IDRs play an important, yet hitherto uninvestigated, role in autophagy. We confirm disorder of selected IDRs via biophysical methods, and use additional bioinformatics tools to predict protein-protein interaction and phosphorylation sites within IDRs, identifying potential biological functions. We experimentally investigate four distinct BECN1 domains: (i) The IDR, which includes a functional BCL2 homology 3 domain (BH3D) that binds BCL2 proteins, undergoing a binding-associated disorder-to-helix transition and enabling BCL2s to inhibit autophagy. (ii) The flexible helical domain (FHD) which has an unstructured N-terminal half and structured Cterminal half forming a 2.5-turn helix in our 2.0 Å X-ray crystal structure. Our molecular dynamics simulations and circular dichroism spectroscopy analyses indicate the FHD transiently samples more helical conformations and likely undergoes a binding-associated disorder-to-helix transition. We also show that the FHD bears conserved residues critical for AMBRA1 interaction and for starvation-induced autophagy. (iii) A coiled-coil domain (CCD) which forms an antiparallel homodimer in our 1.46 Å X-ray crystal structure. We have also built a atomistic model of an optimally packed, parallel BECN1:ATG14 CCD heterodimer that agrees with our experimental SAXS data. Further, we show that BECN1:ATG14 heterodimer interface residues identified from this model are important for heterodimer formation and starvation-induced autophagy. (iv) A β-α repeated autophagy-specific domain which bears invariant residues that we show are important for starvation-induced autophagy. Thus, we demonstrate that conformational flexibility is a key BECN1 feature. Lastly, we show that multi-domain BECN1 constructs have extended conformations with no intra-domain interactions that impact structure of other domains, suggesting that BECN1 structure and conformational flexibility enable its function as an autophagy interaction hub.
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    The Discovery of a Novel Growth Hormone Receptor and the Nutritional Regulation of the Growth Related Actions of Growth Hormone
    (North Dakota State University, 2017) Walock, Chad Napoleon
    The growth hormone (GH) family peptides such as GH, prolactin (PRL), and somatolactin (SL) regulate a wide array of physiological actions including but not limited to growth, metabolism, osmoregulation, and lipolysis. These actions are regulated by many factors both internal and external. I used rainbow trout (Oncorhynchus mykiss) as a model organism to study the effects of GH-family peptides, nutritional state, and serum on insulin-like growth factor (IGF) and growth hormone receptor (GHR) expression. Gene sequencing and phylogenic analysis was applied to characterize a novel GHR. Real-time quantitative-PCR was used to determine IGF and GHR expression levels in liver, muscle, and adipose tissue. Western blotting and pharmacological inhibitors were used to determine signaling pathways. A novel GHR was characterized and determined to be a type 1 GHR with a diverse distribution. It was found to have many features conserved in other GHRs including binding regions, a Y/FGEFS motif, cysteine residues, and N-glycosylation sites. Fasting was shown to decrease GHR1 expression in the liver, adipose tissue and red muscle. GH and PRL were shown to stimulate IGF expression through the ERK, PI3K/Akt, and JAK-STAT signaling pathways. GH-stimulated IGF expression was dependent on nutritional state, as GH was only able to stimulate IGF expression in fed fish. Nutritional state has no direct effect on GH-stimulated GHR expression. Serum was determined to be the mediator of the change in GH sensitivity as pre-treatment with serum from cells of an opposite nutritional state caused cells to react like the opposite nutritional state in GH-stimulated IGF expression. These findings contribute to the understanding of the actions of GH-family peptides and the mechanisms through which GH conducts its diverse actions in times of differing nutritional availability.
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    Nutritional Regulation of Growth Hormone-Stimulated Lipolysis
    (North Dakota State University, 2014) Bergan-Roller, Heather Elaine
    Growth hormone (GH) regulates several physiologic processes in vertebrates, including the promotion of growth, an anabolic process, and mobilization of stored lipid, a catabolic process. Here, we used rainbow trout (Oncorhynchus mykiss) as a model to examine the nutritional programming required for the disparate metabolic actions of GH, specifically lipolysis. Juvenile trout were exposed to fed and fasting regimens in vivo and subsequent hormone treatment in vitro. We used real-time quantitative-PCR to measure levels of mRNA expression of Hormone-sensitive lipase 1 (HSL1) and HSL2 in liver, muscle, and adipose tissue. We used Western blotting to investigate the signaling pathways affected by nutritional state and activated by GH (e.g., JAK-STAT, MAPK, PI3K-AKT, PKC-PLC). In vivo, fasting retarded growth and activated lipolysis through enhanced HSL mRNA expression and protein activation. Moreover, fasting resulted in phosphorylation of ERK and PKC but not Akt, JAK2, and STAT5 in adipose tissue, liver, and muscle. In vitro, GH stimulated glycerol release, HSL mRNA expression, and HSL phosphorylation in a time- and concentration- related manner but only in hepatocytes isolated from fasted and not fed fish. Moreover, these actions were dependent upon PKC-PLC and MAPK-ERK activation but not JAK-STAT or PI3K-Akt action. Nutritional state, insulin, and insulin-like growth factor I (IGF-I) pretreatments affect lipolytic responsiveness in hepatocyte. When in a fed state, with high levels of insulin and IGF, GH links to JAK-STAT pathways to promote growth. In a fasted state, with low levels of insulin and IGF, GH links to lipolysis through PKC and ERK activation. The findings of this dissertation indicate that nutritional status of an organism may mediate the pleiotropic actions of GH by linking it to unique intracellular signaling pathways. In the circumstances of fasting, GH stimulates lipolysis through PKC and ERK activation.
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    Nos1-Adaptor Protein Dysfunction in the Nucleus Tractus Solitarii Contributes to the Neurogenic Heart Damage and Qt Interval Prolongation
    (North Dakota State University, 2015) Singh, Neha
    Variants of the Nitric Oxide Synthase 1 Adaptor Protein (NOS1AP) locus are strongly related to QT interval prolongation and sudden cardiac death (SCD) in human. Neurogenic cardiac damage due to subarachnoid hemorrhage, stroke, epilepsy and myocardial infarction is known to contribute to sudden death in most cases. Our aim was to study the role of NOS1AP in the neurogenic cardiac damage by silencing NOS1AP expression in the Nucleus Tractus Solitarii (NTS) area of the brainstem using lentiviral vector-mediated NOS1AP shRNA (Lv-NOS1AP-shRNA). Real time PCR data showed NOS1AP mRNA levels were expressed in the NTS 3-fold higher than other organs such as kidney and heart in Sprague Dawley (SD) rats. Microinjection of Lv-NOS1AP-shRNA in the NTS caused significant reduction in NOS1AP expression in SD rats. NOS1AP knockdown in NTS did not alter blood pressure (BP), heart rate (HR) recorded by radiotelemetry. However, ECG analysis revealed heart rate variability (HRV) was significantly reduced (SDNN, 51.2±5.6 vs 5.0±1.3ms, P< 0.001, n=6) and QTc interval was markedly prolonged (72.4±4 vs 105±11 ms, P< 0.05, n=6) in NOS1AP knockdown rats. Myocardial damage was also observed with the downregulation of NOS1AP in the NTS of SD rats due to the presence of contraction band necrosis. To study the cellular mechanisms underlying NOS1AP action, we investigated the effect of NOS1AP knockdown on NMDA-induced neurotoxicity in primary cultured neuronal cells from the brainstem. Treatment of cells with Lv-NOS1AP-shRNA significantly reduced NOS1AP expression, and was associated with increased NO production and NMDA-induced neurotoxicity, suggesting a protective effect of NOS1AP. Co-immunoprecipitation studies revealed that the association between neuronal Nitric Oxide Synthase (nNOS) and NMDA Receptor (NMDAR) was significantly increased in neurons treated with Lv-NOS1AP-shRNA, suggesting that NOS1AP might compete with NMDAR in binding to nNOS. Therefore, knockdown NOS1AP expression results in increased association between NMDAR and nNOS, leading to elevated glutamate-induced NO production and neurotoxicity. In summary, all results indicate that NOS1AP plays an important role in the protection of neurons from glutamate-induced neurotoxicity. NOS1AP dysfunction in the NTS might increase the risk of neurogenic cardiac damage, leading to QT interval prolongation, even sudden cardiac death.
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    Effects of Environmental Estrogens on the Growth Hormone-Insulin-Like Growth Factor System in Rainbow Trout (Oncorhynchus Mykiss)
    (North Dakota State University, 2013) Hanson, Andrea M.
    The increasing production, use, and disposal of an expanding array of chemicals that enter the environment pose a serious threat to terrestrial and aquatic animals, as well as to humans. Fish in aquatic habitats are exposed to increasing concentrations of environmental contaminants, including environmental estrogens (EE). In this work, rainbow trout were used to assess the effects of EE on the growth hormone (GH)-insulin-like growth factor (IGF) system, specifically focusing on osmoregulation, organismal growth, and growth at the molecular level. Juvenile trout were exposed to varying concentrations of 17â-estradiol (E2), â-sitosterol (âS), and 4-n-nonylphenol (NP) in vivo and in vitro. Real-time quantitative-PCR was used to measure levels of mRNA expression (GH receptor 1 (GHR1), GHR2, IGF-1, IGF-2, IGF receptor 1A (IGFR1A), and IGFR1B) in multiple tissues, including liver, gill, and muscle. Western blotting was used to elucidate signaling pathways affected by EE-treatment (e.g., JAK-STAT, MAPK, PI3K). Environmental estrogen-treated fish displayed depressed growth in terms of body mass and body length. The observed effects on organismal growth appeared to be due to a decrease in food conversion, as food consumption was not significantly different between treatment groups. Hepatic, gill, and muscle levels of mRNAs encoding GHR1, GHR2, IGF-1, IGF-2, IGFR1A, and IGFR1B decreased in a concentration-, time-, and compound-dependent manner in vivo and in vitro. Furthermore, EE-treated fish displayed decreased osmoregulatory function when subjected to a salt water challenge, as evaluated by measuring plasma chloride levels and mRNA expression of GHRs, IGFs, and IGFRs. The suppression of mRNA expression of components of the GH-IGF system by EE was linked to suppressed phosphorylation of JAK-STAT, MAPK, and PI3K-Akt in a concentration- and time-dependent manner in hepatocytes and gill filaments, an effect that was ER-dependent. Classically, the ER has been thought to function as a nuclear receptor; however, the observed results support the notion that the ER (and thus EE) may have nongenomic effects as well. The results of this dissertation indicate that EE suppress growth at the organismal and molecular level via inhibition of growth-related signaling cascades and repression of gene expression elements of the GH-IGF system.
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    Augmented Expression of Apelin/APJ in the Paraventricular Nuclei of Rats after Myocardial Infarction
    (North Dakota State University, 2012) Pingili, Ajeeth Kumar
    Heart failure (HF) is a disease condition in which insufficient blood is pumped through the body. The pathophysiology of HF is multisystematic and includes a collection of different responses to compensate for the inability of the heart to pump the blood with the most important outcome being increased sympathetic nervous system (SNS) activity. Increased SNS activity leads to reclaim the reserved cardiac function. However, this adaptive response is short term and deleterious. The central mechanisms that lead to increased SNS activity during conditions of HF remain enigmatic. APJ, a G-protein-coupled receptor and its endogenous ligand, is a novel neuroendocrine system. Previous studies from us and others indicated that central administration or over expression of apelin in brain cardiovascular regulatory areas resulted in an increase in blood pressure, sympathetic nerve activity and cardiac hypertrophy. The main objective of this study is to determine whether the Apelin/APJ system is involved in increased SNS activation during HF. We created HF rat models by left coronary artery ligation. Apelin and APJ receptor mRNA levels were measured in cardiovascular regions of the brain of sham and myocardial infarction (MI) rats. Results showed a significant increase in the levels of Apelin/APJ mRNA levels in paraventricular nuclei (PVN) and rostral ventrolateral medulla (RVLM) in MI rats as compared to sham rats. To determine the functional role of elevated APJ receptor in these cardiovascular regulatory regions of the brain during HF, we constructed a lentiviral vector carrying an APJ shRNA (Lenti-APJ-shRNA) to knockdown the APJ receptor. Efficiency of the lentiviral vector to knockdown the APJ receptor was confirmed in vitro by transducing a Cath.a cell line and a primary neuronal cell culture with Lenti-APJ-shRNA. In order to determine the effect of silencing of the APJ receptor in vivo, Lenti-APJ-shRNA virus was injected into the PVN of the MI and sham rats. Results showed knockdown of APJ receptor improved left ventricular function and decreased myocardial fibrosis and hypertrophy in MI rats. Thus, this study shows that PVN plays an important role in sympatho excitation and pathophysiology of HF and these findings may help in developing effective therapies for HF.
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    Immunomodulatory Role of B Lymphocytes and Hyaluronic Acid in a Murine Model of Allergic Asthma
    (North Dakota State University, 2012) Ghosh, Sumit
    In the world today, asthma affects more than 235 million people. The widespread prescription of inhaled corticosteroids—the current gold standard of asthma control medication—allows many asthmatics to live symptom-free and has significantly reduced the number of deaths due to asthma. However, when the disease is poorly controlled, for example due to ubiquitous exposure to airborne fungal conidia, this chronic inflammatory disease often results in lung dysfunction caused by airway architectural changes. The role of B lymphocytes in allergic asthma has been relegated to the production of IgE with relatively little being known about the trafficking of these cells in the tissues or their role(s) in the affected tissue. As a first step in ascertaining their function, the initial aim of this project was to characterize the recruitment and localization of B cells in the murine lung in response to Aspergillus fumigatus inhalation. We found that CD19+CD23+ B2 lymphocytes were recruited to the lungs after fungal inhalation and that IgA-, IgE-, IgG-producing cells localized around the large airways. The second aim of the project was to begin defining the impact that these B lymphocytes have on the allergic lung. By using mice that were deficient of conventional B cells, we were able to demonstrate that the allergic phenotype was retained, although the impact of tissue B1 B cells cannot yet be ruled out. We then investigated the ability of hyaluronic acid (HA), a major component of the extracellular matrix (ECM) generated at sites of chronic inflammation, to recruit and modulate B lymphocyte functions in allergic fungal disease. We found that B lymphocytes undergo chemotaxis in response to LMM HA, while HMM HA had little to no effect on B cell chemotaxis. Furthermore, HA-mediated B lymphocyte chemotaxis was significantly inhibited by blocking the CD44 HA receptor. We also demonstrated that LMM HA fragments elicit the production of the pro-fibrotic cytokines IL-10 and TGF-β1 by B lymphocytes. These observations suggest a previously unrecognized role for B lymphocytes and HA in the context of allergy and represent novel pathways by which B lymphocytes may contribute to airway inflammation and airway remodeling.
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    The Role of Platelet-Derived Growth Factor Receptor Signaling in Medulloblastoma Metastasis
    (North Dakota State University, 2013) Bhat, Kruttika Narayan
    Medulloblastoma is the most common brain tumor in children and one third of the patients remain incurable. Tumor metastasis is one of the primary reasons for its high mortality rate. Despite evidence of overexpression of PDGFRα and PDGFRβ in metastatic medulloblastoma, their individual roles remain controversial and equivocal. Analysis of their specific signaling pathway in medulloblastoma cells revealed that PDGFRα and PDGFRβ signaling events lead to distinct cellular functions: while PDGFRβ stimulated cell proliferation and invasion, the expression of CD44 to regulate progression via c-Myc and inhibited cell death, PDGFRα displayed the opposite effects. Studies also revealed that c-Myc plays an intermediary role by regulating the downstream molecules in PDGFRβ signal pathway such as CD44 and NFB. NFB activity was found to be down- regulated in the absence of PDGFRβ pathway, with its activity restored by the overexpression of c-Myc. Analysis of medulloblastoma patient tissues without a prior knowledge of their metastatic nature further confirmed that PDGFRβ-CD44 axis regulate medulloblastoma metastasis. Co-inhibition studies performed by simultaneous inhibition of both PDGFRβ and c-Myc either by using siRNAs or by using pharmacological inhibitors demonstrated an enhanced inhibitory effect on medulloblastoma cell proliferation and migration. Using miRNA profiling of Daoy cells lacking either PDGFRβ or c-Myc alone or both, a set of miRNAs regulated by both PDGFRβ and c-Myc in common were identified. Integrative analysis of these miRNAs and their targets revealed that activation of PDGFRβ signaling and overexpression of c-Myc may enhance medulloblastoma progression via modulating the expression of several miRNAs such as miR-1280, -1260 and consequently regulating the expression of oncogenic molecules, such as Jagged 2 and CDC25A, respectively. Specific inhibition of miRNAs, miR-1280 and -1260, and JAG2 demonstrated their vital roles in medulloblastoma cell proliferation and migration. These findings suggest that the PDGFRβ-CD44 is a regulatory axis modulating medulloblastoma progression via c-Myc and targeting PDGFRβ/c-Myc/CD44 may provide a novel therapeutic strategy for the treatment of metastatic medulloblastoma.
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    Regulation of Insulin- and Insulin Receptor-Encoding mRNAS in Rainbow Trout, Oncorhynchus Mykiss
    (North Dakota State University, 2012) Caruso, Michael Alexander
    In this work, rainbow trout were used as a model system to examine the regulation of insulin (INS)- and insulin receptor (IR)-encoding mRNA expression profiles. INS- and IR-encoding mRNAs were isolated, cloned, and sequenced; and shown to be differentially expressed within and among multiple tissue types. Regulation was examined through various nutritional and hormonal treatments (in vivo and in vitro). A real-time quantitative-PCR assay was developed to measure the respective levels of mRNA expression. Fasting, growth hormone (GH), and somatostatin (SS) differentially regulated INS and IR mRNAs within selected tissues, in vivo. Glucose, GH, SS, and insulin-like growth factor-1 (IGF-I) differentially regulated INS and IR mRNAs within selected tissues, in vitro. The results of this dissertation research display the identification and differential regulation of multiple INS- and IR-encoding mRNAs and suggest that independent mechanisms may serve to regulate the various isoforms in a tissue-specific manner. Future studies are also suggested.
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    Anticancer Mechanisms of Flaxseed and its Derived Mammalian Lignan Enterolactone in Lung
    (North Dakota State University, 2017) Chikara, Shireen
    Whole flaxseed and its derived lignans have shown anti-cancer properties in a variety of malignancies. However, their potential remains uninvestigated in lung cancer, the leading cause of cancer-related deaths worldwide. We investigated the anti-tumor effects of flaxseed-derived mammalian lignan enterolactone (EL) in human lung cancer cell cultures and the chemopreventive potential of 10% whole flaxseed in a mouse model of lung carcinogenesis. We found that EL inhibits in vitro proliferation and motility of a panel of non-small cell lung cancer cell (NSCLC) lines. EL-mediated inhibition in lung cancer cell proliferation was due to a decrease in mRNA and protein expression levels of G1-phase cell cycle promoters and a simultaneous increase in mRNA and protein expression levels of p21WAF1/CIP1, a negative regulator of the G1-phase. Similarly, EL decreased lung cancer cell motility by modulating cytoskeleton organization, inhibiting the activation of the FAK-Src-paxillin signaling cascade, and expression of down-stream motility regulators. Our in vivo investigation revealed that 10% whole flaxseed reduced the incidence, number, and size of lung tumor nodules in A/J mice exposed to the tobacco smoke carcinogen, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). RNA sequencing revealed altered expression of genes whose products modulate inflammation and oxidative stress, and are likely to be responsible for chemopreventive potential of whole flaxseed. The results from our in vitro studies highlight the anticancer potential of EL in lung cancer, while the results from our in vivo study show that whole flaxseed holds promise as a chemopreventive dietary agent in lung.
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    Mechanisms of Piperlongumine-Induced Cancer Cell Death
    (North Dakota State University, 2015) Dhillon, Harsharan
    Piperlongumine (PPLGM), a bioactive agent obtained from long pepper plants, possesses potent antitumor activity by inducing reactive oxygen species (ROS). However, the mechanisms for PPLGM’s antitumor actions are not well defined. We investigated PPLGM’s antitumor effects and molecular mechanisms against pancreatic and colon cancer, two of the leading causes of cancer death for both men and women in the U.S. We found that PPLGM activated a ROSmediated DNA damage pathway that lead to pancreatic cancer cell death in vitro. Further, mice treated with PPLGM showed reduced pancreatic tumor volume, which was associated with a decrease in tumor cell proliferation and enhanced oxidative stress levels. To elucidate the target pathways responsible for PPLGM-mediated cell death, RNA sequencing was performed. 684 genes were differentially expressed in pancreatic cancer cells treated with PPLGM compared to the control. Genes related to ER-stress and UPR pathways were activated in PPLGM-treated pancreatic cancer cells. To determine the therapeutic efficacy of PPLGM in combination with currently used chemotherapy in vivo, an orthotopic mouse model of pancreatic cancer was used. The combination of PPLGM with gemcitabine resulted in greater reduction of tumor weight and volume than either agent alone, and PPLGM-treated mouse tumors showed decreased expression of Ki-67, a proliferation marker. In vitro studies supported the in vivo results where the combination of PPLGM with gemcitabine and erlotinib significantly decreased pancreatic cancer cell viability and survival, and induced apoptosis compared to control cells or cells treated with the chemotherapeutic agents alone. PPLGM inhibited the growth of colorectal cancer cells to a greater degree than normal colon cells and activated p-ERK protein expression. The use of a MEK inhibitor attenuated the activation of p-ERK and partially blocked PPLGM-mediated cell death, indicating the involvement of the MEK/ERK pathway in colon cancer cell death. These results suggest PPLGM holds potential as a therapeutic agent to treat pancreatic and colon cancer in the clinics.