Cell and Developmental Biology Commons^™

The Effects Of Prenatally Administered Phytoestrogens On The Reproductive And Behavioral Development Of Long-Evans Rats, Crystal Blake

Theses and Dissertations

Equol is known to be a selective androgen modulator and has the ability to bind and inhibit 5-alpha dihydrotestosterone (5α-DHT). Equol is also a selective estrogen receptor modulator and is able to bind beta estrogen receptors with high affinity. As estrogen receptors are found in the hypothalamus, pituitary, and gonads, prenatally administered equol could affect the morphological and reproductive development of offspring. To test this hypothesis, during gestational days 14 to 20, forty-two pregnant Long-Evans rats were given one of six treatments: 1) no treatment, 2) injection with dimethyl sulfoxide (DMSO), 3) injection with 10 mg/kg equol, 4) injection with …

Cholinergic Neurons Regulate And Utilize Gdnf Secreted By C2c12 Skeletal Muscle Cells In Culture, John-Mary Vianney, John Spitsbergen

Biological Sciences Faculty and Graduate Student Research

Glialcell line-derived neurotrophicfactor (GDNF) has been regarded as a potent survival factor for a subpopulation of neurons. It has been shown that GDNF expression is upregulated in skeletal muscle of patients with early stage of neuromuscular diseases such as amyotrophic lateral sclerosis (ALS). Previous results from our laboratory showed that neural cells regulate GDNF secretion by skeletal muscle; non-innervated skeletal muscle appear to secrete more GDNF compared to innervated skeletal muscle. Two aims were examined in the current study. First, to examine whether neural cells inhibit GDNF through acetylcholine release. Second was to examine whether differentiated NG108-15 neural cells secrete …

Thermodynamic Evidence That Ganglioside-Mediated Insertion Of Botulinum A Into The Cholinergic Nerve Ending May Precede Endocytosis And Acidification: A Langmuir Film Study, Bradley Adam Strongin

Theses and Dissertations

Botulinum Neurotoxin (BoNT) is one of the most potent toxins known to human kind. The Atomic Force Microscope (AFM) was employed to investigate the conditions under which BoNT type A heavy chain would bind and/or insert into mica supported dipalmitoylphosphatidylcholine (DPPC) lipid bilayers. As an alternate technique, DPPC/GT1b or total ganglioside extract (80:20) monolayers of a Langmuir Blodgett (LB) Trough were adapted to be artificial membrane models for toxin insertion studies. We conclude that LB monolayer studies are a promising candidate for BoNT/A membrane insertion investigation. Botulinum neurotoxin serotype A insertions into the LB monolayers in the presence of BoNT/A …

Effects Of Endurance Training On The Ampk Response To Exercise., David Gerald Chesser

Theses and Dissertations

Activation of AMP-activated protein kinase (AMPK) results in the upregulation of several intracellular systems which help to prepare a cell for a high energy challenge. The magnitude of the AMPK response to a 10 min bout of exercise has been found to decrease in red quadriceps (RQ) following training, while putative AMPK roles seem to be maintained; specifically, the biogenesis of mitochondria and higher levels of hexokinase II and glucose transporter 4 (GLUT4). If the AMPK response to exercise is responsible in part for these adaptations, how can they be maintained if the AMPK response is attenuated? The purpose of …

Mapk Survival Signaling In Melanoma, Matthew W. Vanbrocklin

Dissertations

Extracellular signals activate mitogen-activated protein kinase (MAPK) cascades potentiating biological activities such as cell proliferation, differentiation, and survival. Constitutive activation of MAPK signaling pathways is implicated in the development and progression of many human cancers, including melanoma. Mutually exclusive activating mutations in NRAS or BRAF are found in ∼85% of all melanomas resulting in constitutive activation of the MAPK pathway (RAS-BRAF-MEK-ERK-RSK). We have previously demonstrated that inhibition of this pathway with small molecule MEK inhibitors selectively induces apoptosis in human melanoma cells both in vitro and in vivo, but not in normal melanocytes. These results support the notion that the …

Maternal High-Salt Diet During Pregnancy In Sprague Dawley Rats Programs Exaggerated Stress-Induced Blood Pressure And Heart Rate Responses In Adult Female Offspring, Summer Hayes King

Theses and Dissertations

The prenatal environment has been shown to have lasting effects on cardiovascular health. In the present study, pregnant rats were fed a 0.7% NaCl normal salt (NS) diet or an 8% NaCl high salt (HS) diet throughout pregnancy. Adult offspring were fitted with radiotelemetry probes to continuously measure blood pressure and heart rate. Rats were placed in restraining cages to test for a programmed acute stress hyperresponsiveness. Offspring were challenged with HS diet for one week to determine if blood pressure salt sensitivity had been programmed by the prenatal HS diet. Animals were killed following resting and acute stress conditions, …

Amp-Activated Protein Kinase Kinase Activity And Phosphorylation Of Amp-Activated Protein Kinase In Contracting Muscle Of Sedentary And Endurance Trained Rats, Denise Hurst

Theses and Dissertations

This study was designed to examine activity of AMP-activated protein kinase kinase (AMPKK) and AMP-activated protein kinase (AMPK) in muscles from control (C) and endurance trained (T) rats. Rats were trained 5 days/wk, 2 hr/d for 8 wks at a final intensity of 32 m/min up a 15% grade with 30 second sprints at 52 m/min every 10 min. Gastrocnemius muscles were stimulated in situ in T and C rats for 5 min at frequencies of 0.4/sec and 1/sec. Gastrocnemius LKB1 protein, a putative component of the AMPKK complex (LKB1, STRAD, and MO25), increased approximately 2-fold in response to training. …

Regulation Of Adrenal Steroidogenesis By Interleukin-6, Stephen A. Mcilmoil

Theses and Dissertations

Cortisol and dehydroepiandrosterone (DHEA) are steroids produced by the zona fasciculata (ZF) and reticularis (ZR), respectively, of the adrenal cortex. Both steroids are upregulated in response to adrenocorticotropic hormone (ACTH). Cortisol is a glucorticoid that is important in the regulation of inflammation and metabolism. DHEA is an adrenal androgen important in fetal growth and puberty but tends to decrease gradually after puberty in both men and women. DHEA has various effects on metabolism and immune function including inhibiting the effects of cortisol on some tissues. During the acute phase of stress, cortisol and DHEA rise due to an increase in …

Regulation Of Lkb1-Strad-Mo25 Complex Expression And Activation Of Ampk In Skeletal Muscle By Thyroid Hormone, Devon Jack Branvold

Theses and Dissertations

AMP-activated protein kinase (AMPK), a heterotrimeric protein which serves as a metabolic master switch in skeletal muscle, is a research target for the pharmaceutical treatment and prevention of type 2 diabetes. The expression of all of the isoforms of the subunits of AMPK and AMPK activity are increased in skeletal muscle tissue of hyperthyroid rats. Activity of AMPK is regulated by an upstream kinase (AMPKK). The LKB1-STRAD-MO25 complex is a major AMPKK in skeletal muscle. This experiment was designed to determine whether the increase in AMPK activity is accompanied by a thyroid hormone-induced increase in the expression of the LKB1-STRAD-MO25 …

The Role Of Stat And The Jak/Stat Pathway In Mediating The Effects Of Interleukin-6 On Star Expression, Janae Strickland

Theses and Dissertations

Cortisol, a hormone produced by a hormone produced by the adrenal gland, is responsible for many regulatory functions in the body. Cortisol release is mediated by adrenocorticotrophic hormone, or ACTH, through the hypothalamus-pituitary-adrenal or HPA axis. This HPA axis is the major release pathway used during acute stress, during which the levels of ACTH parallel those of cortisol. However, in states of chronic stress, the level of ACTH drops dramatically, while cortisol remains high. This study focuses on the pathway of cortisol release during these chronic stress states, specifically examining the role of IL-6 with respect to STATs and the …

Avpv Neurons Containing Estrogen Receptor-Beta In Adult Male Rats Are Influenced By Soy Isoflavones, Edwin D. Lephart, Lihong H. Bu

Faculty Publications

Background Isoflavones, the most abundant phytoestrogens in soy foods, are structurally similar to 17beta-estradiol. It is known that 17beta-estradiol induces apoptosis in anteroventral periventricular nucleus (AVPV) in rat brain. Also, there is evidence that consumption of soy isoflavones reduces the volume of AVPV in male rats. Therefore, in this study, we examined the influence of dietary soy isoflavones on apoptosis in AVPV of 150 day-old male rats fed either a soy isoflavone-free diet (Phyto-free) or a soy isoflavone-rich diet (Phyto-600). Results: The occurrence of apoptosis in AVPV was examined by TUNEL staining. The incidence of apoptosis was about 10 times …

Following Temperature Stress, Export Of Heat Shock Mrna Occurs Efficiently In Cells With Mutations In Genes Normally Important For Mrna Export, Christiane Rollenhagen, Christine A. Hodge, Charles N. Cole

Dartmouth Scholarship

Heat shock leads to accumulation of polyadenylated RNA in nuclei of Saccharomyces cerevisiae cells, transcriptional induction of heat shock genes, and efficient export of polyadenylated heat shock mRNAs. These studies were conducted to examine the requirements for export of mRNA following heat shock. We used in situ hybridization to detect SSA4 mRNA (encoding Hsp70) and flow cytometry to measure the amount of Ssa4p-green fluorescent protein (GFP) produced following heat shock. Npl3p and Yra1p are mRNA-binding proteins recruited to nascent mRNAs and are essential for proper mRNA biogenesis and export. Heat shock mRNA was exported efficiently in temperature-sensitive npl3, yra1 …

Expression And Function Of Alpha3 And Beta2 Neuronal Nicotinic Acetylcholine Receptor Subunits In Hek-293 Cells, Nathan W. Steinhafel

Theses and Dissertations

Single-cell real-time quantitative RT-PCR was used to characterize the mRNA expression of rat neuronal nicotinic acetylcholine receptor (nAChR) subunits α3 and β2 in CA1 hippocampus stratum radiatum and stratum oriens interneurons. α3β2 co-expression was detected in 43% of interneurons analyzed. The nAChR subtype α3β2 was transiently expressed in cells derived from the human embryonic kidney cell line 293 at mRNA levels found in the CA1. The functional properties of α3β2 in HEK-293 cells were characterized by whole-cell patch clamping using acetylcholine (ACh) as an agonist. The kinetics of α3β2 channels were further analyzed by altering the level of α3 DNA …

The Influence Of Membrane Lipid Order On Cell Shape And Microvesiculation In Human Erythrocytes, Laurie Jackson Gonzalez

Theses and Dissertations

Exposure of human erythrocytes to elevated intracellular calcium causes alterations in cell shape and stimulates shedding of the cell membrane in the form of microvesicles. We hypothesized that both the shape transition and microvesiculation are influenced by microscopic membrane physical properties such as lipid order. To test this hypothesis, membrane properties were manipulated by varying the experimental temperature, membrane cholesterol content, and the internal ionic environment. Changes in membrane order were assessed using steady-state fluorescence spectroscopy with an environment-sensitive probe, laurdan. Our observations led us to the following conclusions: 1) the modest temperature dependence of membrane structure observed with laurdan …

Fgf4 And Wnt5a/Pcp Signaling Promote Limb Outgrowth By Polarizing Limb Mesenchyme, Keri Lynn Low

Theses and Dissertations

The focus of this study was to elucidate the molecular and cellular mechanisms whereby fibroblast growth factors (FGFs) mediate outgrowth of the limb. Specifically, we examined the epistatic relationship between FGF and Wnt/Planar cell polarity (PCP) signaling in establishing cell polarity as a mechanism for outgrowth. By implanting beads into embryonic limbs and lateral plate mesoderm, we established that FGF activates Wnt5a in a gradient fashion. Once it was established that Wnt5a was expressed at the right time and place to turn on PCP signaling, we investigated the ability of Wnt5a to influence cell migration and/or cell polarity. Our analysis …

Construction Of A Col11a1 Transgene Vector, Cameron Mckell Beck

Theses and Dissertations

Background: Cartilage disorders affect millions of people in the United States alone, with effects ranging from poor skeletal development and joint pain to shortened lifespan and perinatal lethality. Many of these disorders have their root in defects of collagen, type XI collagen being among the most important. A mouse model of such a type XI collagen defect is the chondrodysplasia (cho) mutant. Mice homozygous for this null mutation in the Col11a1 gene do not express the α1 chain of type XI collagen. This results in a functional knockout of type XI collagen, leading to insufficient skeletal development and perinatal lethality. …

Pka As An Upstream Kinase For Lkb1/Strad/Mo25, Seth Taylor Herway

Theses and Dissertations

The LKB1/STRAD/MO25 complex (LSMK) has been identified as the major upstream kinase for AMP-activated protein kinase (AMPK). PKA phosphorylates LKB1 at the Ser428 residue in humans and Ser431 residue in mice. We investigated PKA as an upstream kinase for LSMK. LKB1 that had been incubated with PKA prior to incubation with AMPK experienced up to a 51% increase in AMPK Kinase activity compared to LKB1 alone (p < 0.05). When blocked with a PKA Inhibitor, the kinase effect of PKA on LKB1 was eliminated. Rat epitrochlearis muscle tissue incubated with epinephrine experienced no increase in AMPK activity compared with controls indicating that epinephrine does not cause AMPK activity in this type of tissue. In conclusion, phosphorylation by PKA can increase the AMPKK activity of LKB1-STRAD-MO25 in vitro. Because LKB1 has been found to be constitutively active, it is postulated that phosphorylation by PKA may act to enhance LKB1-AMPK interaction and thus achieve its effect.

The Effects Of 3-Phosphoglycerate And Other Metabolites On The Activation Of Amp-Activated Protein Kinase By Lkb1/Strad/Mo25, William John Ellingson

Theses and Dissertations

Skeletal muscle contraction results in the phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an upstream kinase, AMPKK. The LKB1-STRAD-MO25 complex is the major AMPKK in skeletal muscle; however, LKB1-STRAD-MO25 activity is not increased by muscle contraction. This relationship suggests that phosphorylation of AMPK by LKB1-STRAD-MO25 during skeletal muscle contraction may be regulated by allosteric mechanisms. In this study we tested an array of metabolites including glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose 1,6-bisphosphate (F1,6-P2), 3-phosphoglycerate (3PG), glucose-1-phosphate (G1P), glucose-1,6-bisphosphate (G1,6-P2), adenosine diphosphate (ADP), carnitine (Carn), acetyl-carnitine (Acarn), inosine monophosphate (IMP), inosine, and ammonia for allosteric regulation. We found that …

Molecular Analysis Of The Effects Of Atrazine On Xenopus Laevis Frogs, Ronald Edward Celestine

Dissertations

Atrazine, the most commonly used herbicide in the U.S., has been implicated as one of the factors that may be causing the worldwide decline of amphibians. First, I examined effects of atrazine on laboratory raised Xenopus laevis tadpoles, treated from 5 days post fertilization to NF stage 65/66 with a concentrationof 25 ppb of atrazine. This study showed no significant difference in body weight, length and metamorphic rate.

Second, I used DNA microarrays to examine mRNA expression levels of genes in atrazine treated and control Xenopus laevis tadpoles. I found significant changes in gene expression levels of 18 genes. These …

Properties Of Conductance And Inhibition Of Proton Channels: M2 From Influenza A Virus And Fo From Escherichia Coli Atp Synthase, Jeffrey C. Moffat

Theses and Dissertations

Proton channels are essential for many of the processes of life. The influenza A viral protein M2 is responsible for sensing the conditions necessary for viral RNA release. The proton-translocating FoF1 ATPase (ATP synthase) uses a proton gradient to drive adenosine triphosphate (ATP) synthesis. We have directly measured proton uptake in vesicles containing reconstituted M2 or FO by monitoring external pH after addition of valinomycin to vesicles with 100-fold diluted external [K+]. This proton flux assay was utilized to quantify proton flux through single M2 and Fo channels. Contrary to previous reports, proton uptake by M2 was not significantly altered …

The Study Of Nitric Oxide Synthase Expression, Function, And Regulation In The Renal Vasculature During Postnatal Renal Development, Brian Blake Ratliff

Theses and Dissertations in Biomedical Sciences

The newborn kidney is vulnerable to vasomotor acute renal failure (ARF) from adverse perinatal events or complications of prematurity. Nitric oxide (NO) vasodilation is vitally protective in this type of ARF, but its relationship with other vasoactive factors, such as angiotensin II (AII) has not been examined. In the immature kidney, nitric oxide synthase (NOS) isoforms, specifically eNOS and nNOS, are developmentally regulated, but their specific role and regulation are unknown.

The enhanced vasodilatory role of NO in the immature kidney was hypothesized to be attributed to regulatory, expressional, and functional differences in eNOS and nNOS isoforms from the adult. …

The Role Of Fgfr4 In Trigeminal Placode Cell Development, Stephanie Beth Reynolds

Theses and Dissertations

In vertebrate embryos, the ophthalmic trigeminal (opV) placode contributes sensory neurons to the trigeminal ganglion during development of the peripheral nervous system. FGFR4 is expressed transiently in the trigeminal placode as cells undergo an epithelial-mesenchymal transition and begin to migrate toward the condensing ganglion. Because of the restricted spatiotemporal expression of FGFR4 in the opV placode, it was hypothesized that FGFR4 is necessary for the process of epithelial-mesenchymal transition in the opV placode. Using electroporation, an FGFR4 inhibitory gene construct was introduced into 6—10 somite stage chick embryos. This secreted inhibitory form of the FGFR4 gene blocked endogenous FGFR4 signaling, …

The Role Of Pax3 In Neuronal Differentiation Of The Ophthalmic (Opv) Trigeminal Placode And Neural Tube During Chicken Embryonic Development, James R. Bradshaw

Theses and Dissertations

Pax3 has been used as a valuable marker in research aimed at understanding tissue interactions involved in trigeminal ophthalmic (opV) placode development. While Pax3 expression coincides with opV neuron specification, the function of Pax3 in these cells has not previously been investigated. Splotch mutant mice (which lack Pax3) have a reduced trigeminal ganglion; however it is not clear whether this reduction is due to neural crest or placode cells. We have used electroporation in the chick model system to block or ectopically express Pax3 at key times in opV placode development. Using several markers of placode cell differentiation, we have …

Influences Of Dietary Soy Isoflavones On Metabolism But Not Nociception And Stress Hormone Responses In Ovariectomized Female Rats, Edwin D. Lephart, Lihong H. Bu, Kenneth Setchell

Faculty Publications

Isoflavones, the most abundant phytoestrogens in soy foods, are structurally similar to 17beta-estradiol. Few studies have examined the nociception and stress hormone responses after consumption of soy isoflavones. Methods: In this study, ovariectomized (OVX) female Long-Evans rats were fed either an isoflavone-rich diet (Phyto-600) or an isoflavone-free diet (Phyto-free). We examined the effects of soy isoflavones on metabolism by measuring body weights, food/water intake, adipose tissue weights as well as serum leptin levels. Also, circulating isoflavone levels were quantified. During chemically induced estrous, nociceptive thresholds were recorded. Then, the animals were subjected to a stressor and stress hormone levels were …

Aging Affects Stretch-Induced P70s6k And 4e-Bp1 Phosphorylation In Fast- And Slow-Twitch Muscle, Sreevani Uddemarri

Theses, Dissertations and Capstones

In the present investigation we compare the expression, basal activation and the ability of muscle stretch to activate the p70^S6k pathway in the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus of adult (6 mo. old), aged (30 mo. old) and very aged (36 mo. old) Fischer 344 x Brown Norway rats. Immunoblotting demonstrated that the tissue content of mTOR, p70^S6k, 4E-BP1 and GSK-3β decreased in the EDL and soleus in aged rats, while SHP-2 increased in the EDL and decreased in the soleus when compared to adult rats. Basal phosphorylation of 4E-BP1 increased in both the …

Dietary Isoflavones Alter Regulatory Behaviors, Metabolic Hormones And Neuroendocrine Function In Long-Evans Male Rats, Edwin D. Lephart, James P. Proter, Lihong H. Bu, Trent D. Lund, Kenneth Setchell, Gina Ramoz, William R. Crowley

Faculty Publications

Phytoestrogens derived from soy foods (or isoflavones) have received prevalent usage due to their 'health benefits' of decreasing: a) age-related diseases, b) hormone-dependent cancers and c) postmenopausal symptoms. However, little is known about the influence of dietary phytoestrogens on regulatory behaviors, such as food and water intake, metabolic hormones and neuroendocrine parameters. This study examined important hormonal and metabolic health issues by testing the hypotheses that dietary soy-derived isoflavones influence: 1) body weight and adipose deposition, 2) food and water intake, 3) metabolic hormones (i.e., leptin, insulin, T3 and glucose levels), 4) brain neuropeptide Y (NPY) levels, 5) heat production …

Phosphorylation Of Skeletal Muscle Acetyl-Coa Carboxylase By Ampk Enhances Palmitoyl-Coa Inhibition, Dustin S. Rubink

Theses and Dissertations

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malnoyl-CoA, which in turn controls the rate of fatty acid metabolism. ACC beta or 2 has been shown to be localized on the mitochondria in close proximity to carnintine palmitoyl transferase 1 (CPT-1), the enzyme responsible for the influx of acyl-CoA into the matrix where beta oxidation takes place. CPT-1 is inhibited by malonyl-CoA produced by ACC. It has been well documented that AMP activated kinase (AMPK) when activated phosphorylates and inactivates ACC. ACC is controlled allosterically by citrate, which activates, and by palmitoyl-COA, which inhibits. In this study, we asked the question, …

The Caenorhabditis Elegans F-Box Protein Sel-10 Promotes Female Development And May Target Fem-1 And Fem-3 For Degradation By The Proteasome, Sibylle Jager, Hillel T. Schwartz, H. Robert Horvitz, Barbara Conradt

Dartmouth Scholarship

The Caenorhabditis elegans F-box protein SEL-10 and its human homolog have been proposed to regulate LIN-12 Notch signaling by targeting for ubiquitin-mediated proteasomal degradation LIN-12 Notch proteins and SEL-12 PS1 presenilins, the latter of which have been implicated in Alzheimer's disease. We found that sel-10 is the same gene as egl-41, which previously had been defined by gain-of-function mutations that semidominantly cause masculinization of the hermaphrodite soma. Our results demonstrate that mutations causing loss-of-function of sel-10 also have masculinizing activity, indicating that sel-10 functions to promote female development. Genetically, sel-10 acts upstream of the genes fem-1, fem-2, and fem-3 and …

Thermal Adaptation Of Maternal And Embryonic Phenotypes In A Wide Spread Lizard (Sceloporus Undulatus), Christopher E. Oufiero

All-Inclusive List of Electronic Theses and Dissertations

Geographically widespread ectotherms encounter thermal gradients that generate variation in behavior, physiology and life-history. Because rates of physiological processes are very sensitive to temperature, cold environments are thought to favor genotypes that have relatively high capacities for growth despite environmental constraints. Two different adaptive responses can occur: (1) mothers in colder environments can allocate more energy to each offspring, or (2) offspring in colder environments make more efficient use of available resources. In this study, I examined both maternal adaptations and embryonic adaptations to environmental temperature. Evidence of maternal adaptations was assessed by examining the relationship between egg size and …

Mechanism Governing The Cellular Susceptibility To Secretory Phospholipase A2, Lauren Blackburn Jensen

Theses and Dissertations

Secretory phospholipase A2 (sPLA2) is an important part of apoptosis and disposal of damaged and dying cells. However, healthy cells are not susceptible to attack by sPLA2. Recent studies have focused on membrane properties necessary to induce susceptibility in both artificial and biological membranes. Hydrolysis of phospholipids by sPLA2 requires at least two preliminary steps: first, adsorption of the enzyme to the cellular membrane, and second, movement of a phospholipid into the active site of the enzyme. We determined the effects of susceptibility on each of the two steps and determined the contributions changing the equilibrium constants have on susceptibility. …