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Theses and Dissertations

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Browsing Theses and Dissertations by Author "Amberg, Gregory C., committee member"

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    Age-dependent decline in Kv4 channels, underlying molecular mechanisms, and potential consequences for coordinated motor function
    (Colorado State University. Libraries, 2019) Vallejos, Maximiliano Jose, author; Tsunoda, Susan, advisor; Amberg, Gregory C., committee member; Bouma, Gerrit, committee member; Mykles, Donald, committee member; Tamkun, Michael, committee member
    The voltage-gated potassium channel, Kv4, is widely expressed in the central nervous system and it is responsible for a highly conserved rapidly inactivating A-type K+ current. Kv4 channels play a role in the regulation of membrane excitability, contributing to learning/memory and coordinated motor function. Indeed, recent genetic and electrophysiological studies in Drosophila have linked Kv4 A-type currents to repetitive rhythmic behaviors. Because a deterioration in locomotor performance is a hallmark of aging in all organisms, we were interested in examining the effects of age on Kv4/Shal channel protein. In this dissertation, I use Drosophila as a model organism to characterize an age-dependent decline in Kv4/Shal protein levels that contributes to the decline in coordinated motor performance in aging flies. Our findings suggest that accumulation of hydrogen peroxide (H2O2) is amongst the molecular mechanisms that contribute to the age-dependent decline of Kv4/Shal. We show that an acute in vivo H2O2 exposure to young flies leads to a decline of Kv4/Shal protein levels, and that expression of Catalase in older flies results in an increase in levels of Kv4/Shal and improved locomotor performance. We also found that the scaffolding protein SIDL plays a role in maintaining Kv4/Shal protein levels and that SIDL mRNA declines with age, suggesting that an age-dependent loss of SIDL may also lead to Kv4/Shal loss. In behavioral studies, we found that a knockdown of SIDL resulted in a lethal phenotype, leading to a large decline in Drosophila eclosion rates, an event that requires coordinated peristaltic motions. Expression of SIDL or Kv4/Shal in this SIDL knockdown genetic background resulted in a partial rescue; these results are consistent with a model in which SIDL and Kv4/Shal play a role in coordinated peristaltic motions and are required for successful eclosion. The results presented in this dissertation provide new insight into the possible molecular mechanisms that underlie an age-dependent decline in Kv4/Shal protein. We identify two contributing factors: 1) ROS accumulation, and 2) the interacting protein SIDL. Our data also suggests that this age-dependent decline in Kv4/Shal levels is likely to be conserved across species, at least in some brain regions. Because Kv4/Shal channels have been implicated in the regulation of long-term potentiation and in repetitive rhythmic behaviors, the loss of Kv4/Shal may contribute to the age-related decline in learning/memory and motor function.
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    Amino acid transmitters and the neural control of feeding and energy homeostasis
    (Colorado State University. Libraries, 2016) Dicken, Matthew S., author; Hentges, Shane T., advisor; Tamkun, Michael M., committee member; Amberg, Gregory C., committee member; Tjalkens, Ronald B., committee member
    Consuming the correct number of calories to maintain a healthy bodyweight is a delicate balancing act between intake and energy expenditure, and humans in modern society seem to have a keen knack for throwing the balance off-center. In the U.S. alone, more than 1/3 of adults are obese based on the body mass index scale, and $147 billion is the estimated annual medical cost for obesity in the United States. On the other end of the feeding spectrum, anorexia in the U.S. has been steadily rising since the 1960s, and has the highest mortality rate of any mental illness. While great strides have been made in understanding the neuronal regulation of energy balance, there is a need to more fully understand the homeostatic systems within the hypothalamus that are so powerful that they are able to drive individuals to poor health or death, often even in the face of consciously fighting their urges. Two groups of functionally opposed neurons contained within the arcuate nucleus of the hypothalamus, Neuropeptide Y / Agouti-related peptide (NPY/AgRP) and proopiomelanocortin (POMC) cells (the so-called first order feeding neurons), have been extensively studied for their roles in energy homeostasis—mostly through research into the peptides they are named after. There is clear evidence that peptides play an essential role for the function of NPY/AgRP and POMC cells, but what about simple amino acid transmitters? While it is known that GABA is packaged and released by NPY/AgRP cells and that this release is relevant to feeding behavior, there is still a dearth of information about this aspect of the circuitry, very much an area waiting to be mined. This study focuses on better understanding the functional release and relevance of amino acid transmitters packaged in both NPY/AgRP and POMC cell populations. Evidence is presented here for the conclusive release of both GABA and glutamate from POMC cells within intact circuitry. For NPY/AgRP neurons, evidence is presented for a shift in functional release of GABA from these neurons onto POMC cells depending on feeding state, corroborated by concurrent in situ hybridization experiments. Using a combination of electrophysiology and in situ hybridization approaches, evidence is also provided that mRNA levels of glutamate decarboxylase can act as a general proxy for functional GABA release. Altogether, these results indicate that amino acid transmitters play a significant role in first order feeding neuron physiology. Not only does this warrant further study on the significance of each transmitter alone and their purpose in comparison with the peptides released, but also the interplay between POMC cell and NPY/AgRP cell amino acid transmitters and their many shared downstream targets. Imbalances in proper glutamatergic and GABAergic signaling may significantly contribute to obesity, and advancing this area of study could lead to correcting those imbalances to restore healthy energy homeostasis.
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    Influence of FADS2 expression on cardiovascular risk: role of mitochondrial arachidonic acid
    (Colorado State University. Libraries, 2020) Li Puma, Lance Christopher, author; Chicco, Adam J., advisor; Bouma, Gerrit J., committee member; Amberg, Gregory C., committee member; Gentile, Christopher L., committee member; Legare, Marie E., committee member
    Long-chain polyunsaturated fatty acids (LC-PUFA) are widely believed to influence cardiovascular health and disease in humans and can be supplied through the diet or endogenously synthesized from the essential PUFAs linoleic acid (LA; n6) and alpha-linolenic acid (ALA; n3). Redistribution of PUFAs in serum and tissue phospholipids has been associated with various pathologies, manifesting primarily as a proportional loss of the essential PUFA LA paralleled by reciprocal increases in its long-chain product arachidonic acid (AA; n6). Epidemiological studies have linked greater AA/LA ratios in serum phospholipids to multiple parameters of cardiometabolic disease (CMD), such as obesity, insulin resistance, hypertension, atherosclerosis, and coronary artery disease. Single nucleotide polymorphisms in the FADS2 gene are associated with haplotypes of greater expression of FADS2, which may increase the production of AA from LA and increase serum AA/LA ratios. FADS2 encodes delta-6 desaturase, the rate-limiting enzyme in endogenous LC-PUFA biosynthesis, which is believed to participate in the development of CMD by interacting with the high dietary LA content found in the modern Western diet to disproportionally produce AA over other LC-PUFAs. The overarching hypothesis of this dissertation is that greater expression of FADS2 promotes the development of cardiovascular risk parameters. To investigate this, we generated mice with global (CMV promoter) transgenic overexpression of Fads2 (Fads2TG); these mice exhibit classic serum shifts in PUFA distribution characteristic of human FADS2 polymorphisms. A series of three projects were undertaken: 1) Investigate the interaction between dietary essential fatty acid intakes and Fads2 expression on cardiovascular risk; 2) Establish methodology for simultaneous measurement of mitochondrial respiration and ROS release in vitro; 3) Investigate effects of Fads2 expression on cardiac mitochondrial responses to Ca++-overload. These studies discovered that greater Fads2 expression is sufficient to increase several aspects of cardiovascular risk that were independent of the dietary ratio of n6:n3 essential PUFAs. Further investigation demonstrated that cardiac cardiolipin AA content predicted ischemia-reperfusion (IR) injury, suggesting a mechanistic role of mitochondria in this phenotype. Gain- and loss-of-function approaches in mice established that greater Fads2 expression lowers mitochondrial tolerance to Ca++-overload demonstrated by loss of OXPHOS-linked respiration, greater mitochondrial ROS release, and increased mitochondrial permeability transition. Furthermore, mitochondrial permeability transition by Ca++-overload could be attenuated by inhibition of AA release or metabolism. Collectively, the significance of these studies establish the influence of Fads2 on serum and tissue PUFA composition and the pathogenesis of IR injury through modulation of mitochondria membrane composition, thereby demonstrating Fads2 expression as an independent factor for cardiovascular risk.
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    Structure-function relationships underlying GluA2 mechanisms of deactivation, desensitization, and modulation
    (Colorado State University. Libraries, 2013) Harms, Jonathan E., author; Partin, Kathryn M., advisor; Amberg, Gregory C., committee member; Prasad, Ashok, committee member; Tamkun, Michael M., committee member
    Glutamate is the primary excitatory neurotransmitter in the central nervous system, where it is principally responsible for mediating excitatory neurotransmission. Ligand-gated receptors to glutamate, such as the a-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptor, are responsible for many cognitive processes; with the AMPA receptor showing an essential role in learning, memory, and synaptic plasticity. As many mental illnesses and diseases show underlying cognitive complications, therapeutic drugs that can alleviate these cognitive deficits show tremendous potential benefit. However, despite great interest and continued advancement, progress of drugs through clinical trials into available treatments has been slow and problematic. One potential reason for the slow progress of drug development is a lack of basic understanding for how compounds bind to AMPA receptors and upregulate their function. Presented here are several studies aimed to better understand how structural interactions regulate AMPA receptor mechanisms of gating and modulation. These studies combine fast-perfusion electrophysiology capable of simulating synaptic events with structural information obtained from x-ray crystallography studies to analyze potential mechanisms of allosteric modulation. Promisingly, we have identified potential patterns relating modulator properties such as size and rigidity with their observed physiological effects. Such patterns suggest that information from these studies can facilitate design of more targeted and efficacious cognition enhancing drugs. In addition to this drug analysis, we identify a new potential drug target site: the AMPA receptor outer vestibule near the ion-conducting pore. We further characterize that alteration to this site acts independently of other modulators, providing a site for modulators that may accompany current pharmacological therapies. Together, these studies demonstrate that structural information can be successfully applied to the process of drug design, with the added benefit of enhancing our understanding for molecular mechanisms of AMPA receptor function.
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    TRPM4 in cerebral artery smooth muscle cells
    (Colorado State University. Libraries, 2012) Gonzales, Albert Louis, author; Earley, Scott, advisor; Amberg, Gregory C., committee member; Dinenno, Frank A., committee member; Rash, John E., committee member; Tamkun, Michael M., committee member
    Cerebral arterial tone is dependent on the depolarizing and hyperpolarizing currents regulating membrane potential and governing the influx of Ca2+ needed for smooth muscle contraction. Several ion channels have been proposed to contribute to membrane depolarization, but the underlying molecular mechanisms are not fully understood. In this review, we will discuss the historical and physiological significance of the Ca2+-activated cation channel, TRPM4, in regulating membrane potential of cerebral artery smooth muscle cells. As a member of the recently described transient receptor potential super family of ion channels, TRPM4 possesses the biophysical properties and upstream cellular signaling and regulatory pathways that establish it as a major physiological player in smooth muscle membrane depolarization.