Larry (Lawrence) Reynolds - Thesis Committee

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Quantifying Seasonal Variation in Physiological Condition of Adult Franklin's Gull (Luecophaeus Pipixcan) during Nesting
(North Dakota State University, 2011) Weissenfluh, Shawn Edward
Understanding seasonal variation in adult physiological condition is important for developing hypotheses on how nest initiation, adult condition, chick development and recruitment are related in Franklin's gull and other migratory species of the northern plains. The purpose of this study was to profile physiological condition during the breeding season in nesting Franklin's gull (Luecophaeus pipixcan) adults. Physiological condition was quantified in nesting adults through four metrics: body measurements recorded from live-trapped birds, the corticosterone stress response measured from blood samples collected serially from live-trapped birds, and two measures of immune function (antimicrobial capacity of plasma from blood samples and heterophil/lymphocyte ratios based on blood smears, both taken from live-trapped birds). Physiological condition declined across the breeding season, as shown by a decline in body condition, stress tolerance and immune performance. Specifically, residual body mass decreased and exposure of the sternum keel increased with the progression of the breeding season. Additionally, birds nesting later in the season showed greater maximum corticosterone concentrations in the stress profile along with lower antimicrobial capacity. These results suggest two hypotheses: 1) that timing of nesting has a significant impact on the physiological condition of Franklin's gull and 2) that birds in poorer condition initiate breeding later in the season. Seasonal variation in condition may be related to time constraints observed in temperate latitudes and whether these birds are capital (i.e., acquiring resources outside the breeding area) or income (i.e., acquiring resources locally) breeders. Thus, determining physiological condition during the breeding season is an important step in elucidating how nest initiation, adult condition, chick development and recruitment are related in Franklin's gull.
Genetic Disruption of VIP Signaling Alters Intestinal Microbial Structure and Immunity
(North Dakota State University, 2018) Bains, Manpreet
Vasoactive intestinal peptide (VIP) regulates clock gene expression in the brain that synchronizes diurnal feeding behaviors in mammals. In the gastrointestinal (GI) tissues, VIP influences host nutrient absorption from ingested food, and regulates host metabolic functions. VIP signaling ensures efficient nutrient absorption by influencing ghrelin and leptin expression to balance caloric intake. Importantly, obese humans have elevated plasma VIP levels, supporting its association with fat mass accumulation. In contrast, VIP deficiency leads to weight loss and reduced adiposity, while disrupting epithelial cell nutrient absorption, tight junctions and mucus secretion. Moreover, VIP regulates host glucose metabolism as VIP knockout mice are pre-diabetic with elevated blood glucose and insulin levels. In addition to metabolism, VIP is anti-inflammatory and when knocked out results in exacerbated inflammatory bowel disease (IBD) pathology. The GI track is also home to ≈40 trillion bacteria, called the gut microbiota, which unlock additional calories from fiber for the host. Microbiota dysbiosis is caused by dysfunction in biological systems downstream from VIP signaling, including dysregulated expression of host clock genes, metabolic hormones, immune-relevant mediators and metabolic and inflammatory disease states, like obesity and IBD. It is not known, however, whether the VIP signaling axis contributes to the maintenance of the gut microbiota structure and diversity. We hypothesized that VIP deficiency will cause gut dysbiosis, lower bacterial diversity and reduce its energy extraction potential. To this end, we isolated fecal samples from VIP knockout mice (VIP-/-) and employed 16S rRNA sequencing. VIP deficiency (VIP-/- and VIP-/+) resulted in marked gut microbial compositional changes and reduced bacterial diversity compared to male and female VIP+/+ littermates (n=48). Increased abundance of Bacteroides, Parabacteroides and Helicobacter genera (gram-negative, GN), with reductions of Lachnospiraceae NK4A136, Oscillibacter and Ruminiclostridium genera (gram-positive, GP), were the driving force for the observed increase in the GN/GP ratio. A predicted algorithm program, called PICRUSt, showed changes in microbial metabolism consistent with elevated lipopolysaccharide metabolism and reduced intake of fiber in VIP-/- mice. These data support that VIP regulates intestinal homeostasis by maintaining microbiota balance, diversity and energy harvesting potential, while upholding an anti-inflammatory tone by limiting lipopolysaccharide biosynthesis.
Biological Evaluation of the Associations Between Animal Size, Feeding Behavior, Blood Metabolites and Feed Efficiency in Beef Cattle
(North Dakota State University, 2017) Portela Fontoura, Ananda Barbara
The biological variation in feed efficiency is regulated by multiple physiological mechanisms relevant to energy use in livestock species. The current study examined the associations between body composition, feeding behavior, linear body measurements and plasma metabolites with different measures of feed efficiency in growing heifers, finishing steers and mature pregnant cows. Our findings indicate that inclusion of body size measurements in prediction models of gain and intake improved the models’ accuracy and might account important differences related to eating capacity. Among the traits evaluated, feeding behavior possessed stronger associations with efficiency measures and displayed differences between efficient and inefficient animals. The associations between the traits studied herein varied across the efficiency measures used and beef cattle stage of production. Thus, selection criteria and performance evaluation based on efficiency measures should account for these traits, combined with animal’s stage of production and system’s outputs of interest.
Effects of Maternal Nutrition on Fructose, Glucose, and Cationic Amino Acid Transporter Expression in Bovine Utero-Placental Tissues from Days 16 to 50 of Gestation
(North Dakota State University, 2016) Crouse, Matthew Scott Pennell
Poor Maternal nutrition has been implicated to reduce nutrient transport to the conceptus. Therefore, we hypothesized that maternal nutrition and day of gestation would impact mRNA expression of nutrient transporters GLUT1, GLUT3, GLUT5, GLUT14, CAT-1, CAT-2, and CAT-3 in beef heifers. Crossbred Angus heifers (n = 49) were bred via AI, assigned to nutritional treatment (CON = 100% of requirements for 0.45 kg/d gain and RES = 60% of CON) and ovariohysterectomized on d 16, 34, or 50 of gestation. Expression of CAT-2 was the only gene in any tissue to demonstrate a day × treatment interaction. Expression of nutrient transporters were not influence by nutritional treatment (P > 0.05); however, transporters were differentially expressed by day of gestation (P ≤ 0.05). We interpret these results to indicate that day, has a greater influence than a 40% global nutrient restriction on mRNA expression of nutrient transporters in bovine utero-placental tissues.
The Impact of Digestive Enzymes in the Ruminant Animal
(North Dakota State University, 2016) Keomanivong, Faithe Elizabeth
Understanding the enzymatic activity in ruminant digestive systems is essential for securing adequate growth, reproduction and overall metabolism. In order to evaluate the impact of various nutritional sources and dietary strategies on enzymatic activity, five experiments were designed. Experiment 1 examined the influence of nutrient restriction and melatonin supplementation on maternal and fetal pancreatic development. Experiment 2 explored various phases of the reproductive cycle and the impact of intravenous arginine infusion amid differing levels of feed intake. Experiment 3 determined the effectiveness of realimentation during advancing stages of gestation. Experiments 4 and 5 examined the impact of variable rations on ruminal pH, NH3, VFA, total gas and methane concentration and enzymatic activity in steers consuming rations of fine- vs coarse-rolled corn and 20% vs 40% DDGS (Experiment 4) and corn vs barley based diets with low- vs moderate-oil DDGS (Experiment 5). Overall, nutrient restriction caused reduced BW, pancreatic mass and pancreatic enzyme activity in mature animals. In Experiment 1, the addition of dietary melatonin diminished the impact of nutrient restriction on maternal pancreatic mass and α-amylase activity while reducing the secretion of insulin and size of insulin-containing cell clusters. Fetal pancreatic enzymes were unaffected by treatment, however, pancreatic morphology exhibited greater insulin-containing cell cluster size in fetuses from adequately fed dams. In Experiment 2, arginine infusion did not alter pancreatic exocrine or endocrine function during the various luteal stage phases. In Experiment 3, realimentation during different stages of gestation decreased the impact of reduced feed intake and, in some cases, allowed for compensatory gain of the exocrine pancreas. The maternal and fetal endocrine pancreas was unaffected. Mature animals had greater changes in pancreatic exocrine secretions whereas fetuses differed mainly in endocrine function as a result of improper nutritional status. Comparison of pancreatic tissue revealed a greater quantity, and larger size, of insulin-containing cell clusters in fetuses which appear to separate as the animal matures. Differences in rumen enzymatic activity was found in Experiments 4 and 5, however, despite changes in lag time of gas production or ruminal degradation rates, the concentration of greenhouse gases (CH4 or CO2) produced were unaffected.
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.
Maternal Nutritional Plane and Endogenous Retroviral Gene Elements, Pregnancy Hormones, and Placental Vascularity and Angiogenic Factors during the Establishment of Pregnancy in Beef Cattle
(North Dakota State University, 2016) McLean, Kyle James
In order to meet the projected food demands by 2050, animal agriculture must increase production of animal products on the same or decreased land area through increased efficiency. Early gestation is one area to increase efficiency in beef production in a twofold manner 1) by increasing the number of calves born due to decreased early embryonic loss and 2) by minimizing detrimental effects due to fetal programming which may decrease offspring growth or reproductive efficiency. Both of which will result in more pounds of beef produced by the same number of cows. Recently, endogenous retroviral elements (ERV), which make up a significant portion of mammalian genomes, have been implicated in vital steps during placentation. The placenta is the source of nutrient, gas, and waste exchange between maternal and fetal circulation which is necessary to support fetal growth. Maternal nutrition influences fetal growth and placental development. Therefore, we hypothesized that ERV envelope genes, syncytin-Rum1 and BERV-K1, as well as pregnancy specific hormones, PSP-B, and IFN-τ will be differentially expressed during critical time points of early pregnancy and maternal nutrition restriction will alter mRNA expression at critical time points. We developed a technique to ovariohysterectomized beef heifers which provides a large animal model to acquire uteroplacental tissues. In year 1, we established basal expression patterns for syncytin-Rum1 and BERV-K1, PSP-B, and IFN-τ within utero-placental tissues during the first 50 d of gestation. In year 2, we determined the effects of 40% global nutrient restriction on the mRNA expression of syncytin-Rum1 and BERV-K1, PSP-B, and IFN-τ on d 16, 34, and 50 of gestation in uteroplacental tissues. These data provide novel evidence of differential expression of endogenous retroviruses (syncytin-Rum1 and BERV-K1), PSP-B, and IFN-τ during early gestation but 40% maternal nutrient restriction had little influence of mRNA expression. However, further work needs to be completed to elucidate functions, mechanisms, and interactions of these genes during early gestation and their importance to the successful establishment of pregnancy.
Arginine Supplementation Strategies during Gestation: Impacts on Dams and Offspring
(North Dakota State University, 2015) Bjertness, Jena Lee
We hypothesize rumen-protected arginine supplementation during gestation will mitigate deleterious offspring effects caused by undernutrition. Experiment 1: non-pregnant ewes were supplemented with rumen-protected arginine at varying doses to assess effects on circulating amino acids and carotid hemodynamics. Arginine concentrations post-supplementation were greater in ewes supplemented with 180 vs. 90 mg/kg BW, and vascular resistance indices were lesser with 180 mg/kg BW; therefore, 180 mg/kg BW was used in experiment 2. Experiment 2: nutrient-restricted pregnant ewes were supplemented with rumen-protected arginine and maternal and offspring growth and physiological responses were measured. Arginine supplementation to nutrient restricted ewes improved offspring development compared to restricted ewes without supplementation. Circulating amino acids in offspring were efficiently metabolized, which may contribute to improved growth and development. There was no change in carotid hemodynamics in supplemented pregnant ewes. Further research should determine how arginine improves development, as this dietary supplement could rescue at-risk pregnancies.
Implementation of Fourier Repetitive Control in Labview
(North Dakota State University, 2014) He, Fan
This thesis presents position control as well as current (torque) control of a DC servo motor with a periodic input. Position control is achieved by using Fourier repetitive control scheme. Simulation on position control is carried out and results discussed. Experiment is executed based on a testing platform that is built using two, mechanically linked DC servo motors and LabVIEW to incorporate the Fourier repetitive control algorithms. The experiment results validate the feasibility of controlling motor positions via Fourier repetitive control scheme. Suggestions on improving the implementation of Fourier repetitive control theory are also made. Experiment results on current (torque) control are presented. The controlling of both position and current of the motors at the same time is also demonstrated.
Growth Hormone Mediated Regulation of Osmoregulation in Euryhaline Teleosts
(North Dakota State University, 2014) Martin, Lincoln
Within the multitude of fish species that exist on our planet, there are a certain number that possess the unique ability to live in both freshwater (FW) and seawater (SW) environments. This ability, known as euryhalinity, is limited to a relatively small number of species, thus making it a prime target for scientific research into osmoregulation, due to the uniqueness of this ability. It has been shown previously that growth hormone (GH) plays an important role in regulating this ability, and in this work, Atlantic salmon (Salmo salar) and Rainbow trout (Oncorhynchus mykiss) were used as models to examine the expression of specific osmoregulatory genes in response to SW transfer and GH exposure, and to examine the signaling mechanisms used by GH to facilitate any changes. We found that GH utilizes specific cell signaling pathways to facilitate the transition between FW and SW in both Rainbow trout and Atlantic salmon.
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.
Impacts of Supplemental Arginine on Ewe Reproductive Performance
(North Dakota State University, 2014) Crane, Alison Ryan
The objective of this study was to determine the effects of injectable and oral arginine (Arg) supplementation provided 14 d post-breeding on reproductive performance of fall lambing ewes. Upon estrus detection (d 0) ewes were randomly assigned to one of six treatments for a 14-d treatment period: injectable saline (CON; n = 25), injectable alanine (IVALA; n = 20), injectable arginine (IVARG; n = 23), oral rumen-protected Arg (RPARG; n = 20), oral soybean meal (SBM; n = 23), or oral fishmeal (FM; n = 24). Weaning rates were higher (P < 0.05) in Arg supplemented ewes. Plasma progesterone and serum Arg concentrations exhibited a treatment and day effect (P < 0.05), but no treatment × day interaction was observed (P > 0.05). In contrast to previous research, supplemental Arg during the first 14 d of pregnancy did not improve pregnancy or lambing rates, however, IVARG positively impacted weaning rates.
Effects of Corn Condensed Distillers Solubles Supplementation on Intake, Performance, Rate and Site of Digestion, and Ruminal Fermentation in Cattle Consuming Forage-Based Diets
(North Dakota State University, 2015) Coupe, Lindsey Rebecca
Two studies were conducted to determine effect of feeding method and level of corn condensed distillers solubles supplementation on performance of beef cows fed forage-based diets and effects on digestibility and ruminal fermentation. Experiment 1 utilized 80 gestating crossbred cows in a randomized complete block design. Treatments were arranged in a 2 × 2 + 1 factorial design; main effects were feeding method (mixed vs. fed separately) and level of CCDS. All treatments were offered ad libitum forage. Experiment 2, utilized 5 ruminally and duodenally cannulated Holstein steers in a 5×5 Latin square to evaluate effects of CCDS supplementation on DM intake, site of digestion, and ruminal fermentation. Exp. 2 utilized similar treatments as experiment 1; and all treatments were offered ad libitum forage. Results of these studies suggest that CCDS supplementation increases intake, performance, and CP digestion and appears to be an effective supplement for cattle eating moderate-quality forages.
Biophysical Studies of Ligand Binding to Human Histone Deacetylase-8
(North Dakota State University, 2014) Singh, Raushan Kumar
Due to an involvement in various patho-physiological conditions, human histone deacetylases (HDACs) are high priority drug targets for the treatment of several diseases, such as cancer, heart failure, neurodegeneration, etc. An effector (inhibitor/activator) of these enzymes has a great potential to alleviate the above disease conditions. In this regard, HDAC inhibitors – Zolinza and Istodax - have already been approved by the FDA for the treatment of T-cell lymphoma, aside from several other inhibitors which are in the advanced level of clinical trials. HDAC8 serves as a prototype to study structural-functional and catalytic features of human HDACs. In order to pursue the biophysical studies of the ligand-binding, HDAC8 was cloned, expressed, and purified from E. coli. A high-throughput screening (HTS) of an in-house library of small molecules was performed utilizing a trypsin-coupled in vitro HDAC8 assay to discover novel effectors of HDAC8, and the N-acetylthiourea and the thiopyridine derivatives were discovered as the isozyme-selective inhibitors and activators of HDAC8, respectively. In vitro HDAC8 assay utilizing a fluorogenic peptide as a substrate often produces artifactual results. Therefore, a substrate-independent HDAC8 assay was developed utilizing a fluorescent analog of a pan-HDAC-inhibitor. In view of the fact that the downstream cellular response of a drug is often dictated by the transient kinetic and thermodynamic parameters of its interaction with the target, the transient kinetics and thermodynamics of interaction of the selected HDAC8 inhibitor with the enzyme were thoroughly investigated. It was observed that the dissociation off-rate and/or the enthalpy of binding of an HDAC8 inhibitor to the enzyme could play a crucial role in determining its in vivo efficacy. A rationale has been presented that the above parameters of the ligand-protein interaction could be utilized for optimizing a drug candidate (HDAC8 inhibitor) in order to enhance its in vivo potency.
Impacts of Beef Cow Nutrition on Conceptus Development
(North Dakota State University, 2013) Camacho, Leticia Elideth
Two experiments were conducted to determine the effect of maternal nutrient restriction followed by realimentation during early to mid-gestation on uterine blood flow (BF), maternal performance, and conceptus development in pregnant beef cows. In Experiment 1, effects of maternal nutrient restriction followed by realimentation during mid-gestation on uterine BF of lactating, multiparous cows were evaluated. Nutrient restriction from d 30 until 140 of gestation did not alter total uterine BF. However, upon realimentation (from d 140 to 198 of gestation), there was enhanced ipsilateral uterine BF. In Experiment 2, effects of maternal nutrient restriction followed by realimentation during early to mid-gestation on late gestation uterine BF, maternal performance, and conceptus development was evaluated using non-lactating, multiparous cows. Slaughters were performed at d 85, 140, and 254 of gestation. During late gestation when all cows were receiving similar nutrition (100% of the NRC requirements), ipsilateral uterine BF and total BF were increased in cows that were previously nutrient restricted from d 30 until d 85 of gestation and realimented until d 254 of gestation. Therefore, results from both experiments suggest that the bovine placenta may be programmed to function differently after a period of nutrient restriction. Duration of restriction or realimentation impacted maternal performance and organ weights. The dam might become more efficient in the utilization of nutrients after being realimented as gestation advances. Nutrient restriction during early pregnancy tended to increase fetal and placental size by d 85. However, when cows were restricted longer or when realimented, there were no observable differences in placental or fetal growth. The maternal system may adapt to allow for fetal catch up growth during later gestation by enhancing uteroplacental nutrient transport capacity or placental function. From the results obtained in these 2 experiments we can conclude that maternal nutrient restriction during early gestation enhances conceptus growth and uterine BF later in pregnancy. Perhaps, timely management strategies might result in enhanced conceptus development. Even though more research is necessary, opportunities to intervene appear to be available during times of poor nutrition in beef cow/calf systems.
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.
Influence of Maternal Nutrient Intake on Placental Vascular Function in Pregnant Beef Cows
(North Dakota State University, 2014) Reyaz, Arshi
We hypothesized that global maternal nutrient restriction during early and mid-gestation followed by realimentation in pregnant beef cows would alter placental arterial vascular function. We tested changes in placental caruncular (CAR) and cotyledonary (COT) arterial sensitivity to bradykinin (BK), a potent vasodilator. Cows were randomly assigned to be nutrient restricted for 55 or 110 during early to mid pregnancy. On d 85, 140, and 254 cows were euthanized and CAR and COT arteries were isolated. Maternal nutrient restriction during early and mid-gestation allowed for placental compensation to overcome the loss of nutrients while realimentation returned placental arterial vosoactivity similar to control cows in response to BK. Further, CAR and COT placental arteries may respond to BK induced vasodilation through different pathways which is important when considering possible therapeutics for compromised pregnancies.
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.
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.
An Insilico Design of Nanoclay Based Nanocomposites and Scaffolds In Bone Tissue Engineering
(North Dakota State University, 2016) Sharma, Anurag
A multiscale in silico approach to design polymer nanocomposites and scaffolds for bone tissue engineering applications is described in this study. This study focuses on the role of biomaterials design and selection, structural integrity and mechanical properties evolution during degradation and tissue regeneration in the successful design of polymer nanocomposite scaffolds. Polymer nanocomposite scaffolds are synthesized using aminoacid modified montmorillonite nanoclay with biomineralized hydroxyapatite and polycaprolactone (PCL/in situ HAPclay). Representative molecular models of polymer nanocomposite system are systematically developed using molecular dynamics (MD) technique and successfully validated using material characterization techniques. The constant force steered molecular dynamics (fSMD) simulation results indicate a two-phase nanomechanical behavior of the polymer nanocomposite. The MD and fSMD simulations results provide quantitative contributions of molecular interactions between different constituents of representative models and their effect on nanomechanical responses of nanoclay based polymer nanocomposite system. A finite element (FE) model of PCL/in situ HAPclay scaffold is built using micro-computed tomography images and bridging the nanomechanical properties obtained from fSMD simulations into the FE model. A new reduction factor, K is introduced into modeling results to consider the effect of wall porosity of the polymer scaffold. The effect of accelerated degradation under alkaline conditions and human osteoblast cells culture on the evolution of mechanical properties of scaffolds are studied and the damage mechanics based analytical models are developed. Finally, the novel multiscale models are developed that incorporate the complex molecular and microstructural properties, mechanical properties at nanoscale and structural levels and mechanical properties evolution during degradation and tissue formation in the polymer nanocomposite scaffold. Overall, this study provides a leap into methodologies for in silico design of biomaterials for bone tissue engineering applications. Furthermore, as a part of this work, a molecular dynamics study of rice DNA in the presence of single walled carbon nanotube is carried out to understand the role played by molecular interactions in the conformation changes of rice DNA. The simulations results showed wrapping of DNA onto SWCNT, breaking and forming of hydrogen bonds due to unzipping of Watson–Crick (WC) nucleobase pairs and forming of new non-WC nucleobase pairs in DNA.

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