Life Sciences Commons^™

X-Band Rapid-Scan Epr, Deborah Gale Mitchell

Electronic Theses and Dissertations

The advantages of rapid-scan EPR relative to CW and pulse techniques for samples with long longitudinal relaxation time T₁ (N_s⁰ defects in diamond, N@C₆₀, and amorphous hydrogenated silicon), heterogeneous samples (crystalline 1:1 α,γ-bisdiphenylene-β-phenylallyl (BDPA):benzene), lossy samples (aqueous nitroxyl radicals), and transient radicals (5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO)-superoxide adduct) were studied.

For samples with long relaxation times, CW (continuous wave) EPR is challenging due to power saturation and distortions from passage effects. In rapid-scan EPR, the field is swept through resonance in a time that is short relative to T₂. In rapid-scan EPR, the magnetic field is …

Regulation Of Actin Dynamics During Drosophila Germband Extension, Ashley M. Motlong

Electronic Theses and Dissertations

Convergent extension is a process that occurs in the development of a wide variety of organisms, including gastrulation in the Drosophila embryo to begin to lay out the adult body plan. In fly embryos, this is known as germband extension and is mainly driven by cell intercalation or neighbor exchange by planar polarized cell-cell interface contraction to shorten the tissue along the dorsal-ventral axis. In this thesis, I show that interface contraction consists of phases of fast interface shortening and intervals of stable interface size. My data also suggests that regulation of F-actin aggregates at these shrinking interfaces is important …

Novel Functions For Neuronal Rna Processing Bodies In The Control Of Axon Terminal Growth In Drosophila Melanogaster, Sarala Joshi Pradhan

Electronic Theses and Dissertations

In this thesis we first characterized neuronal functions for HPat/Pat1, a core component of RNA processing bodies or "P bodies". We show that hpat mutants exhibit a strong synaptic hyperplasia at the developing and acutely stimulated Drosophila larval neuromuscular junctions (NMJs). The synaptic defects observed in hpat mutants are associated with rearrangement of the axonal microtubule cytoskeleton suggesting that HPat negatively regulates presynaptic microtubule-based growth during NMJ development. Interestingly, we also found that both pre-and postsynaptic HPat expression controlled rapid axon terminal growth in response to acute spaced synaptic stimulation. We also demonstrate that HPat interacts genetically with the catalytic …

Mitochondrial Glutathione Transport: Implications For Bcl-2 And Neuronal Survival, Heather Marie Wilkins

Electronic Theses and Dissertations

Oxidative stress is a contributing factor to many neurodegenerative diseases. In particular, mitochondria are a key source of oxidative stress due to electron leakage at the level of the electron transport chain. To combat the endogenous production of reactive oxygen and reactive nitrogen species mitochondria are equipped with several redox-cycling systems, such as glutathione (GSH). Mitochondrial GSH has been shown to be a critical reservoir of this antioxidant, where selective depletion of mitochondrial GSH can induce apoptosis in several systems. Many studies have intricately linked Bcl-2 to cellular GSH status and it has been previously shown that Bcl-2 is a …

Seeded Propagation Of Tau Fibrils, Paul David Dinkel

Electronic Theses and Dissertations

In various neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, Pick's disease, and corticobasal degeneration, the deposition of fibrils composed of misfolded tau protein is observed. Recent evidence suggests that tau fibrils transfer between cells and spread throughout the brain, underscoring the significance of fibril propagation.

Six tau isoforms exist in the adult human brain that can be grouped into 4-repeat (4R) tau and 3-repeat (3R) tau based on the presence or absence of the second of four microtubule binding repeats. We demonstrate in vitro that seeded fibril growth, a prerequisite for the spreading of the tau pathology, is crucially …

Evolution Of Melanocortin-2 Receptor Activations: Studies On A Mammal And A Fish, Liang Liang

Electronic Theses and Dissertations

The structure and function relationship between melanocortin-2 receptor (MC2R) and ACTH are the most complicated in melanocortin receptor gene family. A comparative study on the activation of human and rainbow trout MC2R will provide a useful model system for understanding how ACTH emerged as the sole ligand for the MC2R of bony vertebrates. This dissertation will discuss how studies utilizing analogs of hACTH(1-24) have revealed two critical amino acid motifs in this ligand (HFRW and KKRRP) which are required for the activation of MC2R. In addition, the KKRRP motif functioned as the unique binding site for MC2R that directly contributes …

A Role For Dynamin-Dependent Endocytosis During Drosophila Gastrulation, Marissa Kay Kuhl

Electronic Theses and Dissertations

Gastrulation, a process conserved among many higher organisms, is the directed migration of cells into layers that will establish various tissues targeted to become anatomical structures. This process is accomplished through another conserved morphogenetic event, known as cell intercalation. Early in development, this movement of cells within an organized tissue leads to unique cellular arrangements where neighboring cells contract their shared interfaces in order to meet at a shared vertex. In this thesis, I present work that demonstrates a requirement for Dynamin-dependent endocytosis during these contraction events. Using quantitative analysis, I have identified varied cell behaviors during experiments which knockdown …

Isolation And Characterization Of A Full Length Retrotransposon: Cr1, Cassandra Michelle Weason

Electronic Theses and Dissertations

Transposable elements (TE) have been found in all genomes and have clearly had a major impact on genomic evolution. The described research takes advantage of an abundant transposable element present in the genomes of Anseriformes, called Chicken Repeat 1 (CR1). Previous studies in Anseriformes suggest that CR1 is presently active in recent evolutionary time (St. John, 2004). A fully functional CR1 element itself is approximately 4.5kb long (Kajikawa, 1997), where almost all inserts are truncated at the 5' end. Because of this, it has been a challenge to isolate a full length, active element. In this study, two CR1 sequences …

Connexin-32 And Connexin-43 Immunoreactivity In Rodent Taste Buds, Amanda E. Bond

Electronic Theses and Dissertations

Studies indicate that ATP is one of the primary neurotransmitters in taste transduction. ATP release occurs from taste cells via specific hemichannels such as pannexin/connexin hemichannels (Huang et al., 2007; Romanov et al., 2007). We hypothesize that Type II (receptor) and possibly Type III (presynaptic) cells release ATP at sites containing pannexin/connexin hemichannels. In this study, we examine the presence of connexin–32–LIR (Like Immunoreactivity) and connexin–43–LIR in rodent taste buds through immunocytochemical analysis and DAB (Di–amino–benzidine) immunoelectron microscopy. We observed that connexin–32–LIR co–localizes with P2X2–LIR in nerve fibers and in a small subset of NCAM–LIR cells. Connexin–32–LIR does not co–localize …

Analyzing The Interaction Between Melanocortin 5 Receptor, Melanocortin Receptor Accessory Protein 1, And Melanocortin 2 Receptor In Rainbow Trout, Domicinda M. Hill

Electronic Theses and Dissertations

Using CHO cells we sought to explore and characterize the functional relationship of rainbow trout MC5 receptor (rtMC5R) with zebrafish MRAP1 (zfMRAP1) and rainbow trout MC2 receptor (rtMC2R), as well as how the effect of such relationships may play a significant role in Hypothalamic-Pituitary-Adrenal/Interrenal axis activation. This research demonstrated that rtMC5R can be successfully expressed and functionally activated in CHO cells and, in a manner similar to mammals, α-MSH is the preferred ligand for rtMC5R. The presence of MRAP1 does not seem to inhibit the expression or function of rtMC5R, rather it appears that it may increase expression and total …

Fire-Derived Charcoal Along An Ecological Gradient In The Colorado Front Range, Christopher William Licata

Electronic Theses and Dissertations

Terrestrial ecosystems are shaped by natural disturbances such as wildland fire. In the intermountain western United States, forests, shrub and grasslands adapt to repeated fires. An important long-term legacy of wildland fires is black C (BC) commonly referred to as char or charcoal. Black C is a recalcitrant C form that has been long known to influence soil physical, chemical, and biological processes that they vary across landscapes and over time. The objective of this research is to address two key areas in the emerging field of ecosystem BC research; 1) how much BC as charcoal C is formed per …

Structural Polymorphism In Tau Filaments: An Implication For Neurodegenerative Diseases, Ayisha Siddiqua

Electronic Theses and Dissertations

Tau filaments are the pathological hallmark of >20 neurodegenerative diseases including Alzheimer's disease, Pick's disease, and progressive supranuclear palsy. In the adult human brain, six isoforms of tau are expressed that differ by presence or absence of the second of the four semiconserved repeats. As a consequence, half of the tau isoforms have three repeats (3R tau), whereas the other half has four repeats (4R tau).

Site-directed spin labeling of recombinant tau in conjunction with electron paramagnetic resonance spectroscopy was used to obtain structural insights into tau filaments. The studies showed that the filaments of 4R tau and 3R tau …

Reduce, Reuse, Recycle? An Examination Of Factors Influencing Environmental Behavior In Denver, Colorado, Dan J. Kasper

Electronic Theses and Dissertations

Non-recycling and non-composting of municipal solid waste have important natural resource management implications, in that they both reduce energy, water, and raw natural resource use. Responsible waste management also likely has positive climate impacts by virtue of reducing greenhouse gas emissions. Recycling and composting are pro-environmental behaviors that have been shown to be influenced by numerous socio-demographic and psychological factors. This study analyzes the correlation of a number of variables with frequency of recycling and composting in select census tracts in Denver, CO, USA, with the goals of informing waste management policy and contributing to the overall pro-environmental behavior literature. …

Estradiol Modulation Of Calcium Dynamics In Pituitary Mmq Lactotroph Cells, Monika Pauckova

Electronic Theses and Dissertations

Pituitary lactotrophs are excitable cells that exhibit spontaneous, calcium influx triggering prolactin (PRL) secretion to stimulate lactation. Lactotrophs express estrogen receptors (ER) and are a well established estrogen-responsive cell system. 17B-estradiol (E2) is known to directly affect lactotrophs by increasing PRL transcription and biosynthesis, intracellular Ca2+ concentration ([Ca2+]i) and PRL secretion. This study demonstrates that the MMQ clonal cell line, isolated from the 7315a rat pituitary tumor, is a model lactotroph cell line that is E2-responsive. Spontaneous and evoked Ca2+ transients were especially sensitive to L-type channel block, but not affected by block of omega-conotoxin-GVIA-sensitive CaV channels or TTX-sensitive voltage-gated …

Dopamine-Beta-Hydroxylase And Norepinephrine Transporter Immunoreactivity In Rat Taste Buds, Jenna Ashley Walton

Electronic Theses and Dissertations

Of the handful of neurotransmitters modulating taste response, norepinephrine (NE) may be the least understood. Although the physiological role of NE has been tested, it is still unclear whether the rat taste bud may serve as an endogenous source of neurotransmitter or if it must be taken up from outside the taste bud. Immunocytochemical analysis of DBH has shown that DBH–LIR is present in taste cells of the rat circumvallate papillae. Specifically, DBH–LIR is present in a subset of Type II taste cells. Nearly all (98%) DBH–LIR cells express PLCβ2–LIR, and about half (41%) of PLCβ2–LIR cells express DBH–LIR. DBH–LIR …

Cultural Diabetes, Keri E. Noll

Electronic Theses and Dissertations

Diabetes has become a cultural problem across America, but particularly in the Midwest and Southern regions. For my film, I explored these two areas and compared the food and exercise culture to one of the healthier regions, the West. Having moved from Indiana to Colorado, I used my own personal experiences to analyze why people in the Midwest struggle with diabetes and obesity at such a higher rate than those nearer to the Pacific Ocean. Through a series of interviews with close friends and observational analysis of each state, I came to learn something very important about health: our food …

Nutraceutical Antioxidants And Their Therapeutic Potential In Neurodegeneration, Erika Kristine Ross

Electronic Theses and Dissertations

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease that affects motor neurons of the brain and spinal cord. Many studies indicate that mitochondrial oxidative stress (MOS) is a principal mechanism underlying the pathophysiology of this and other devastating neurodegenerative diseases. Here, we investigated a unique whey protein supplement (Immunocal®) to determine its neuroprotective efficacy in several in vitro models of MOS and in an in vivo mouse model of ALS. This non-denatured whey supplement contains cystine which is an oxidized form of cysteine, an essential precursor for synthesis of the endogenous antioxidant, glutathione (GSH). In primary cultured rat cerebellar …

A Hydrodynamic Method For Measuring Aqueous Nanoparticle Surface Interactions, Kellen John Sorauf

Electronic Theses and Dissertations

The objectives of this research dissertation were to develop and present novel analytical methods for the quantification of surface binding interactions between aqueous nanoparticles and water-soluble organic solutes. Quantification of nanoparticle surface interactions are presented in this work as association constants where the solutes have interacted with the surface of the nanoparticles. By understanding these nanoparticle-solute interactions, in part through association constants, the scientific community will better understand how organic drugs and nanomaterials interact in the environment, as well as to understand their eventual environmental fate. The biological community, pharmaceutical, and consumer product industries also have vested interests in nanoparticle-drug …

Pomc Is Expressed In Pancreatic Alpha Cells, Samia M.M Farrara

Electronic Theses and Dissertations

Pancreatic alpha cells secrete glucagon to increase blood glucose during hypoglycemia. Currently, the mechanisms that initiate glucagon secretion are not well understood. This study investigates the expression of pro-opiomelanocortin (POMC), a potent regulator of glucagon secretion, in alpha cells.

Insulin tolerance tests in mice lacking the POMC gene showed a correlation between alpha MSH, and glucagon secretion from alpha cells (Hochgeschwender et al. 2003). Results from a previous study showed that the POMC product, melanocortin á-MSH, acts through a melanocortin receptor 4 (MC4R) at the surface of the alpha cells to trigger the release of glucagon [Angleson, unpublished data, Lumsden …

Mitochondrial Dna Analysis By Denaturing High-Performance Liquid Chromatography For The Characterization And Separation Of Mixtures In Forensic Samples, Richard Kristinsson

Electronic Theses and Dissertations

A mixture of different mtDNA molecules in a single sample is a significant obstacle to the successful use of standard methods of mtDNA analysis (i.e., dideoxy dye-terminator sequencing). Forensic analysts often encounter either naturally occurring mixtures (e.g., heteroplasmy) or situational mixtures typically arising from a combination of body fluids from separate individuals. The ability to accurately resolve and interpret these types of samples in a timely and cost efficient manner would substantially increase the power of mtDNA analysis and potentially provide valuable investigative information by allowing its use in cases where the current approach is limited or fails. Therefore, this …

Sequence Detection And Comparative Analysis Of The Hv1 And Hv2 Control Regions Of Human Mitochondrial Dna By Denaturing High-Performance Liquid Chromatography, Sarah E. Lewis

Electronic Theses and Dissertations

The objective of this research was the evaluation and forensic validation of Denaturing High-Performance Liquid Chromatography (DHPLC) as a sequencingindependent means of detecting the presence of sequence differences in pair-wise mixtures of non-concordant amplicons of human mitochondrial DNA (mtDNA). The reproducibility and efficacy of DHPLC results, including amplification reproducibility, injection reproducibility, and column-to-column reproducibility were measured, showing negligible assay-to-assay variability. In addition, cross-contamination on the DHPLC columns demonstrated very low level DNA carryover between a high-abundance sample and subsequent zero-volume injections.

The accuracy with which DHPLC technology can be used to screen both evidence and control samples in the context …

Camp And Calcium Interactions In Endocrine Cells, Colette Crane

Electronic Theses and Dissertations

Inhibitors of cAMP phosphodiestereses (PDEs) promoted an increase in intracellular [cAMP]_c and an influx of Ca²⁺, in both MMQ pituitary cells and α-Pancreatic cells. The specific targets for PDEs and the domains of PDEs are still unknown. Pituitary MMQ cells were used as a model to test modulation of calcium influx through the L-type calcium channel with PDE inhibitors. cAMP analogs were used to determine cAMP molecular targets. 8-cpt-cAMP initiated an Epac mediated pathway and had strong effects on calcium influx in the cell; suggesting, a relatively strong cAMP target. Experiments of α-Pancreatic cells were done in …

Micrornas 9a, 9b, 9c And 315 Regulate Expression Of A Reporter For The Neuronal Microtubule-Associated Protein Futsch/Map1b, Leslie M. Rozeboom

Electronic Theses and Dissertations

Fragile X syndrome (FXS) is the most common form of inherited mental retardation in humans. FXS is caused by loss of the Fragile X Mental Retardation Protein (FMRP), an important regulator of neuronal mRNA translation. Patients with FXS display cognitive deficits including memory problems. Protein synthesis-dependent long-term changes in synaptic plasticity are involved in the establishment and maintenance of long-term memory. One prevalent theory of FXS pathology predicts that FMRP is required to negatively regulate the translation of important mRNAs at the synapse. We are investigating microRNAs (miRNAs) as a potential regulator of synaptic FMRP-regulated mRNAs that have previously been …

Genetic Polymorphisms And The Treatment Outcome In Standard-Risk Pediatric Acute Lymphoblastic Leukemia, Qi Wei

Electronic Theses and Dissertations

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and accounts for 30 - 35% of all cancers in children. Significant improvement in the treatment of pediatric ALL has been achieved in recent years. Only 50 years ago, the disease was uniformly fatal with an Overall Survival (OS) rate < 5%. Modern-day, multi-drug chemotherapy is associated with an overall survival rate over 80%. Standard-risk ALL comprises the majority of ALL with an overall survival approaching 90%. Despite this success, children who relapse from this disease accounts for the majority of cancer-related deaths in children. The backbone of treatment protocols have incorporated somatic but not host genetic features in the treatment regimens. The current study examined 12 genetic polymorphisms affecting the pharmacodynamics of antileukemic drugs in an attempt to identify biologic markers related to the risk of disease relapse.

In the current research program, 125 standard-risk ALL patients who were treated at The Children's Hospital were enrolled in a retrospective study. Statistical analysis was performed to evaluate the association between genetic polymorphisms and risk of disease relapse in this study cohort. The GSTM1 null genotype was associated with a …

Acute Synaptic Activity Causes Differential Mirna Expression In The Drosophila Melanogaster Larval Central Nervous System, Robert Ian Sand

Electronic Theses and Dissertations

The primary goal of this thesis was to determine if spaced synaptic stimulation induced the differential expression of microRNAs (miRNAs) in the Drosophila melanogaster central nervous system (CNS). Prior to attaining this goal, we needed to identify and validate a spaced stimulation paradigm that could induce the formation of new synaptic growth at a model synapse, the larval neuromuscular junction (NMJ). Both Channelrhodopsin- and high potassium-based stimulation paradigms adapted from (Ataman, et al. 2008) were tested. Once validation of these paradigms was complete, we sought to characterize the miRNA expression profile of the larval CNS by miRNA array. Following attainment …

Functional Activation Of Cartilaginous Fish Melanocortin Receptors, Christina Lee Reinick

Electronic Theses and Dissertations

The evolution of the melanocortin receptor (MCR) gene family has been dictated by two genome duplication events (2R hypothesis). The gnathostomes are thus predicted to possess a minimum of four MCR genes. Previous studies on cartilaginous fish have shown evidence for the presence of only three MCRs. The purpose of this thesis is to functionally express the MC5R from the spiny dogfish (Squalus acanthias) and the putative MC2R and MC3R from the elephant shark (Callorhinchus milii); these receptors have not yet been fully characterized. In this study, SacMC5R was able to be expressed in CHO cells …

Plasma Membrane Dynamics In The Drosophila Embryo, Amelia E. Zommer

Electronic Theses and Dissertations

Convergent extension is a highly conserved process among mammals, in which the tissue narrows in one axis, and extends across another. Tissue elongation is directed by the regulation of cell interface behaviors, which guides cell intercalation and rosette formation. Rosette formation occurs through the contraction of vertically oriented cell interfaces, and the subsequent elongation of new horizontal interfaces. It has been shown that actomyosin-generated tension functions to direct rosette formation. In this thesis, I have tested the function of regulators of F-actin networks, as well as endocytic and exocytic mechanisms, to identify new components that control interface behaviors and cell …

Hepatitis C Virus-Induced Hepatocellular Carcinoma: Cancer Stem Cell And Gene Therapy, Heba Samir Esmaeil Allam

Electronic Theses and Dissertations

Hepatitis C virus (HCV) is an important human pathogen that causes chronic hepatitis cirrhosis, steatosis and hepatocellular carcinoma (HCC) worldwide. Although current FDA-approved drugs are partially effective in the treatment of HCV, methods for cure and vaccine against the virus are still awaited. My thesis work presented here is focused on finding mechanism (s) of HCV-induced hepatocarcinogenesis and devising novel means to combat the HCV infection.

Herein, we have shown that long-term HCV expression results in the acquisition of cancer stem-like cells (CSCs) traits in liver derived cell lines. These traits include enhanced expression of putative stem cell markers DCAMKL-1, …

Memory Access Patterns For Cellular Automata Using Gpgpus, James Michael Balasalle

Electronic Theses and Dissertations

Today's graphical processing units have hundreds of individual processing cores that can be used for general purpose computation of mathematical and scientific problems. Due to their hardware architecture, these devices are especially effective when solving problems that exhibit a high degree of spatial locality. Cellular automata use small, local neighborhoods to determine successive states of individual elements and therefore, provide an excellent opportunity for the application of general purpose GPU computing. However, the GPU presents a challenging environment because it lacks many of the features of traditional CPUs, such as automatic, on-chip caching of data. To fully realize the potential …

Altered Calcium Handling In Cerebellar Purkinje Neurons With The Malignant Hyperthermia Mutation, Ryr1-Y522s/+, George C. Talbott

Electronic Theses and Dissertations

To investigate the etiology of malignant hyperthermia and central core disease, mouse models have recently been generated and characterized (Chelu et al., 2006). These RyRY522S/+ knock-in mutant mice provide an excellent tool to investigate calcium dysregulation, its pathological consequences, and potential therapeutic approaches. Skeletal muscle harboring this mutation exhibits calcium leak from internal stores and an increased sensitivity to activation by caffeine, voltage, and temperature (Durham et al., 2008). Although alterations in RyR1 channel function and resultant changes in cellular function have been characterized in skeletal muscle, the effects of MH mutations in RyR1 on central nervous system function have …