Browsing by Author "Mykles, Donald, committee member"
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Item Open Access 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 memberThe 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.Item Open Access Changing myoglobin's paradigm: characterizing the role between lipids and myoglobin expression(Colorado State University. Libraries, 2014) Schlater, Amber E., author; Kanatous, Shane B., advisor; Florant, Gregory, committee member; Mykles, Donald, committee member; Bell, Christopher, committee memberMyoglobin (Mb) is a muscular heme protein generally localized to oxidative muscle, where it functions to store and transport oxygen, as well as scavenge nitric oxide and reactive oxygen species (ROS). While the former role of Mb in oxygen storage/transport is undisputed in diving mammals and other hypoxia-adapted species, this function appears to be far more ambiguous in terrestrial, non-hypoxia-adapted species. During endurance exercise, terrestrial mammals rely on erythrocytic oxygen to fuel aerobic metabolism in working muscle. Physiological changes associated with endurance training elicit responses that increase muscular blood flow and subsequent oxygen delivery. Intramuscular oxygen stores, alternatively, appear to bear little significance in sustaining aerobic metabolism during endurance exercise, as evident by the inability to appreciably release intra-muscular stored oxygen during normoxic exercise; yet, terrestrial endurance athletes who tend to have a higher reliance on lipid-fueled metabolism have more Mb than their sedentary counterparts. Accordingly, Mb's traditional functional paradigm pertaining to oxygen storage and transport does not appear to be fully applicable to terrestrial mammals in vivo. Here, a series of datasets are provided offering alternative paradigm, where increases in Mb expression are associated with increases in lipid supplementation. C2C12 cells cultured in normoxic and hypoxic (0.5% oxygen) environments show increased Mb when supplemented with a 5% lipid mixture compared to glucose controls. While Mb regulatory pathways have been shown to involve Ca2+ signaling pathways via calcineurin (CN), this lipid-induced Mb stimulation is not affiliated with an increase in CN expression, suggestive of a regulatory pathway for Mb independent of Ca2+. Moreover, lipid-induced Mb stimulation parallels oxidative stress marker augmentation concomitant with Mb augmentation. Addition of antioxidant to lipid-supplemented cells reverses Mb increases, and acute exposure to H2O2 during hypoxic differentiation showed an increase in Mb relative to control cells, collectively suggesting a Mb regulatory pathway through redox signaling. Furthermore, comparison of two commonly used Mb assay techniques revealed that normoxic lipid-induced Mb increases are nearly explicitly oxidized, thus bearing important functional implications on Mb increases consequent of lipid stimulation. In light of these novel data and in conjunction with the inability of terrestrial mammals to appreciably utilize Mb oxygen stores during exercise, an alternative paradigm for Mb is proposed. I propose that the role of Mb as an antioxidant defense during terrestrial exercise, which increases lipid-based aerobic metabolism and ROS production, is more relevant and applicable than a role relevant to storage and transport of oxygen.Item Open Access Gamma-aminobutyric acid (GABA) in the development of the paraventricular nucleus of the hypothalamus (PVN): implications for adult disease(Colorado State University. Libraries, 2012) Stratton, Matthew S., author; Tobet, Stuart, advisor; Amberg, Gregory, committee member; Bamburg, James, committee member; Mykles, Donald, committee memberThe paraventricular nucleus of the hypothalamus (PVN) is the final common regulator of the neuroendocrine stress response. Humans with depression or anxiety disorders display altered regulation of this system and females are more likely to suffer from these disorders than males. This work investigated embryonic development of the PVN to identify cellular processes that might occur incorrectly (preferentially in females) and predispose the individual to altered regulation of stress responses. GABA acts as a neurotrophic factor during development. As the embryonic PVN is ringed by GABA (absence of GABA in the PVN) and a receptor for GABA is enriched in the PVN, it was hypothesized that this molecule would direct PVN development. Embryonic development was altered in mice either by genetic manipulation (receptor knockout mice) or by pharmacological blockade of the GABAA and GABAB receptors. Embryonic GABAA receptor antagonism caused a decrease in the number of neurons that expressed estrogen receptor α in and around the PVN. In female but not male mice lacking GABAB receptors, the cytoarchitecture of the PVN was altered. Specifically, estrogen receptor containing cells were misplaced and corticotropin releasing hormone was increased. Animals treated with a GABAB receptor antagonist during embryonic development copied the phenotype of receptor knockout mice. The in vivo effect of GABA signaling on cell placement was investigated in vitro with organotypic slice fluorescence video microscopy. Again only in females, blockade of the GABAB receptor caused neurons to increase migration speed. Thus GABA acts to restrict cells from moving outside of the PVN. When the GABAB receptor is antagonized, cells migrate outside of the PVN. To determine the consequence of an animal having altered PVN development, animals treated as embryos with the GABAB receptor antagonist were subjected to a battery of behavior tests as adults. Interestingly, females treated embryonically with CGP 55845 displayed an increased anxiety-like phenotype (female specific disorder) while males treated with the same compound displayed a hyperactivity-like phenotype (male specific disorder). Independent of sex, animals treated as embryos with the GABAB receptor antagonist displayed decreased depression-like behaviors and had a less robust stress response compared to vehicle treated animals. This work highlights the importance of GABA signaling in PVN development and the dependence of complex adult behaviors on embryonic brain organization as GABA receptor antagonism limited to a specific critical time period during embryonic development recreated cytoarchitectural and behavioral phenotypes of GABA receptor knockout mice.Item Open Access Inhibition of a truncated form of human mitochondrial kidney-type glutaminase (hKGA124-551) by bis-2-(5-phenylactamido-1,2,4-thialdiazol-2-yl)ethyl sulfide (BPTES)(Colorado State University. Libraries, 2011) Hartwick, Erik William, author; Curthoys, Norman, advisor; Ho, P. Shing, committee member; Peersen, Olve, committee member; Mykles, Donald, committee memberMitochondrial glutaminase (GA) catalyzes the hydrolysis of glutamine producing glutamate and an ammonium ion. There are three isoforms of mammalian GA that are essential to hepatic ureagenesis, renal ammoniagenesis, synthesis of the neurotransmitter glutamate, and the catabolism of glutamine. Here we focus on the human KGA isoform that is predominantly expressed in kidney, brain, intestine, and tissues of the immune system. Recent publications suggest that GA is a novel target for developing new cancer therapeutics. These studies have indicated that inhibition of GA by small molecule inhibitors significantly reduces the size of tumors in rats and inhibits growth of transformed cells in culture. A truncated form of human KGA hKGA124-551 that contains amino acids 124-551, was produced to delete the C-terminal sequences that are unique to the KGA and GAC isoforms. This construct was assayed in the presence of (bis-2-(5-phenylactamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES). BPTES is a potent small molecule inhibitor of mammalian GA that was previously shown to inhibit rat KGA in µM concentrations. In the current study, we adapted the standard GA assay to a microtiter plate format and used it to characterize the inhibition of hKGA124-551 using µM amounts of BPTES. Our data indicate that BPTES is a mixed non-competitive inhibitor at low concentrations of phosphate, but at higher phosphate concentrations the inhibition is predominantly uncompetitive. Lastly, gel filtration and dynamic light scattering experiments were performed to determine if hKGA124-551 oligomers are formed in the presence of BPTES and to characterize the effect of increasing concentrations of phosphate. The data suggest that in low phosphate and in the absence of BPTES, the hKGA124-551 exists as a dimer, but in the presence of BPTES and higher phosphate concentrations the molecular weight shifts to a tetramer or higher oligomer. The combined data indicate that BPTES is a potent lead compound for the development of a therapeutic inhibitor of human GA that may be a potential cancer therapeutic.Item Open Access Molecular regulation of glial inflammation in Parkinson's disease(Colorado State University. Libraries, 2014) De Miranda, Briana R., author; Tjalkens, Ronald, advisor; Kato, Takamitsu, committee member; Legare, Marie, committee member; Mykles, Donald, committee memberParkinson's disease (PD) is the most prevalent movement disorder that affects adults, the primary pathology of which includes the loss of dopamine producing neurons from the substantia nigra (SN) and inflammatory activation of immune mediators in the brain. Reactive astrocytes and microglia have been implicated in driving the progressive phase of dopamine neuron loss in PD, the result of which leads to worsening neurodegeneration over time that no current therapy can inhibit. A significant need exists for small molecule therapeutics that reach the CNS, decrease glial activation, and confer neuroprotection over the progressive phase of PD. A novel class of anti-inflammatory compounds, originally shown to have anticancer properties, are examined here as potential therapeutic agents to decrease glial inflammation in a progressive mouse model of PD. Derived from the condensation product of indol-3-carbinol produced in cruciferous vegetables, para-phenyl substituted diindolylmethanes or C-DIMs have been shown to induce orphan nuclear receptor family NR4A in several different cancer lines, and have reduced inducible nitric oxide synthase (NOS2) expression in primary murine astrocytes. In order to examine the neuroprotective benefit selected C-DIM compounds have on the survival of dopamine neurons during the inflammatory progressive phase of PD, several experimental studies were conducted, and reported here. The initial development of a method using immunofluorescent sereological counts of TH-positive neurons in the SN, resulted in the ability to measure early neuronal degeneration with sensitivity to small changes in neuron loss through design-based 3D modeling of the SN. Additionally, a pharmacokinetic analysis of four C-DIM compounds determined that structural variations changed the metabolism of these compounds in mice, however all C-DIMs tested were orally bioavailable, distributed to the brain, and displayed pharmacokinetic profiles equivalent to current PD therapies. Initial neuroprotective studies demonstrated that three of the selected C-DIMs are able to limit the progression of dopamine neuron loss from the SN after the onset of a lesion, in a progressive PD mouse model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 80 mg/kg) and the drug clearance inhibitor probenecid (250 mg/kg, expressed as MPTPp). A post lesion model was utilized in transgenic NF-κB-EGFP adult mice, where MPTPp treatment was given for 7 days (MPTPp7d) and animals were given either corn oil (vehicle) or one C-DIM (DIM-C-pPhOCH3, DIM-C-pPhOH, DIM-C-pPhCl) via daily oral gavage for the next 7 days (MPTPp14d). Further investigation into the mechanism by which CDIMs reduced dopamine neuron loss revealed that activation of microglia and astrocytes was reduced in the SN from MTPPp7d to MPTP14d in animals given daily oral gavage of the compounds, compared to corn oil control. NF-κB-EGFP intrinsic reporter detection of NF-κB expression was reduced in C-DIM treated animals compared to animals that received no treatment from MPTPp7d to MPTPp14d. Reduction in NF-κB related cytokines (TNF, IL-1α, IFNγ) were also seen after C-DIM treatment. As a final investigation into C-DIM mechanism of action, DIM-C-pPhCl was examined in the BV-2 microglia cell line treated with LPS to stimulate cytokine production. DIM-C-pPhCl treatment reduced the expression of inflammatory proteins such NOS2, TNFα, and IFNγ after LPS treatment, and its effectiveness was dependent on the nuclear receptor transcription factor NR4A2 (Nurr1). In addition, DIM-C-pPhCl treatment increased the amount of Nurr1 present at the NOS2 promoter, suggesting that it enhances the Nurr1-dependent transrepression of NF-κB, recently described as a novel mechanism in glial cells to reduce inflammatory protein and protect against the progression of PD.Item Open Access Physicochemical characterization of self-associated chromatin oligomers(Colorado State University. Libraries, 2016) Rogge, Ryan, author; Hansen, Jeffrey, advisor; Luger, Karoline, committee member; DeLuca, Jennifer, committee member; Mykles, Donald, committee memberThe DNA of chromosomes is extensively compacted within the nuclei of eukaryotic nuclei. Chromosomes are composed of chromatin which is a repeating polymer of nucleosomes bound by additional chromatin proteins. Chromatin can be reconstituted in vitro using purified DNA and histone proteins to form nucleosomal arrays. Reconstituted chromatin fibers are structurally dynamic and the structures formed are highly dependent on the buffer conditions, particularly polyvalent cations. The addition of Mg2+ favors nucleosome-nucleosome interactions. At low concentrations nucleosomes on the same fiber interact resulting in folding, while at higher concentrations inter-fiber interactions result in chromatin self-association. Unlike folded chromatin, the oligomeric structure of chromatin is unkown, to address this deficiency, in this dissertation the oligomeric structures formed by 12-mer nucleosomal arrays were characterized by microscopy, sedimentation velocity, and SAXS experiments. The oligomeric chromatin complexes were globular throughout all stages of the cooperative assembly process, and ranged in size from ~50 nm to a diameter of ~1000 nm. The oligomer sedimentation coefficients under these conditions ranged from 5000-350,000S, corresponding to ~1-400 Mb DNA/oligomer. The nucleosomal arrays were packaged within the oligomers as interdigitated 10-nm fibers, rather than folded 30-nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl2, but became disrupted in 0 mM MgCl2, conditions that also dissociated the oligomers in vitro. These results indicate that a 10-nm array of nucleosomes has the intrinsic ability to self-assemble into large chromatin globules stabilized by nucleosome-nucleosome interactions, and suggest that the oligomers are good in vitro model for investigating the structure and organization of interphase chromosomes.Item Open Access Reexamining the role of linker histones beyond 30 nm fibers in a complex chromatin environment(Colorado State University. Libraries, 2024) Kuerzi, Amanda, author; Hansen, Jeff, advisor; Stargell, Laurie, committee member; Stasevich, Tim, committee member; Mykles, Donald, committee memberEukaryotic cells store DNA in the cell nucleus in the form of chromatin. Chromatin is composed of nearly equal parts proteins and DNA. It is both highly compacted and organized into discrete domains within the nucleus. However, the manner in which chromatin is compacted, and domains are organized, remains elusive. The primary players in chromatin compaction are core histones, which bind DNA to form the nucleosome and the basis for 10 nm fibers. Linker histones also play an important role in chromatin compaction. Previous work showed that linker histones are important for the formation of 30 nm structures. 30 nm structures were long held to be folding intermediates for repressive chromatin domains. However, there is little evidence for these structures in most eukaryotic cell types. Instead, chromatin appears to be composed of an interdigitated 10 nm fibers in both repressive and accessible chromatin types. The role of linker histones in 10 nm fibers is not well characterized. Previous work showed that linker histones stabilized 30 nm structures, rendering them inaccessible to binding by additional proteins. In the following, we investigate the behavior of linker histones in an interdigitated 10 nm fiber environment. We use an in vitro model called "condensates" to mimic the formation of 200 nm chromatin domains. We find that linker histones stabilize these condensates by cross-linking chromatin fibers. Importantly, we show that the presence of linker histones does not preclude binding by additional proteins. Linker histones readily bind condensates in ratios above an expected one linker histone per nucleosome. Additional binding by linker histones suggests that 10 nm fibers provide a complex environment in which linker histones dynamically interact with both nucleosomes and linker DNA.  Item Open Access Temporal examination of myoglobin and myosin heavy chain expression patterns in vitro(Colorado State University. Libraries, 2020) Larson, Ashley Mari, author; Kanatous, Shane, advisor; Hamilton, Karyn, committee member; Mykles, Donald, committee memberMyoglobin is a hemoprotein expressed in vertebrate muscle that is typically known to follow an established oxidative muscle fiber type, found in aerobic muscles. However, more recent evidence has demonstrated changes in myoglobin expression without a change in fiber type, indicating myoglobin expression could be regulated by different pathways and may not always be dependent on a prior expressed fiber type. Myoglobin structure is characterized by a globin backbone that supports a nonprotein heme prosthetic group containing iron, which is responsible for the reversible binding of several ligands such as oxygen, nitric oxide, and carbon monoxide. Naturally, it has been implicated in oxygen transport and storage, nitric oxide and reactive oxygen species scavenging, and cellular lipid transport. Mixed lipid supplementation alone and coupled with hypoxia elevates skeletal muscle myoglobin levels, but it is unknown how these culture treatments affect myoglobin expression relative to the fiber type. Given the uncertainty of when myoglobin is expressed independently from oxidative fiber types, we aimed to determine when differentiating C2C12 cells begin to express myoglobin compared to when they express oxidative isoforms of myosin heavy chain when subjected to factors known to increase myoglobin expression; hypoxia, lipid and/or caffeine treatments. We found that under control and hypoxic conditions, regardless of lipid supplementation, myoglobin expression occurred before oxidative fiber expression. Conversely, cells receiving caffeine stimulation expressed myoglobin following oxidative fiber type expression. Cells exposed to hypoxia and lipid supplementation displayed elevated functional myoglobin expression compared to caffeine stimulated cells, suggesting that this combination of treatments may be more effective at increasing myoglobin than stimulation alone. Overall, the work presented here has determined conditions under which expression of myoglobin precedes oxidative fiber type expression and within these treatments, conditions that also increase functional myoglobin concentration. These findings can act as a step in the process to assist in revealing more about how myoglobin can be expressed in skeletal muscle. Examination of alternate routes of myoglobin expression that are not reliant on prior expression of a particular fiber type could yield potential therapeutic benefits of expressing myoglobin in tissues to combat ischemic diseases seen in humans.Item Open Access The effect of dam nutrient deprivation on lamb carcass characteristics, retail yields, and nutrient composition(Colorado State University. Libraries, 2012) Brenman, Kristina Anne, author; Belk, Keith, advisor; Woerner, Dale, committee member; Engle, Terry, committee member; Mykles, Donald, committee memberThe objective of this study was to determine the effect of dam nutrient restriction on offspring carcass characteristics, retail cut yields, and nutrient composition. Forty one western white rams and ewes were obtained from a previous Colorado State University study of dam nutrient restriction. Prior to gestation, dams were fed 100% of their nutrient requirements. The diet of dams was a vitamin-mineral rich pelleted beet-pulp (77.8% total digestible nutrients [TDN], 90.0% dry matter [DM], and 9.4% crude protein [CP]). At 28 days gestational age, dams were randomly assigned to individual pens and separated into three different treatments: control (100% nutrient requirements), half ration (fed 50% of their nutrient requirements from day 28 until term), and realimented (fed 50% of their nutrient requirements from day 28 until day 78, and then slowly realimented back to 100% for the remainder of gestation). All twin lambs were slaughtered, and hot carcass weight, 12th rib fat, body wall thickness, adjusted fat, ribeye area, ribeye marbling, leg score, leg circumference, conformation, flank streaking, flank firmness, flank color, kidney fat weight, L*, a*, and b* were obtained. After all lambs were slaughtered, one half of each lamb carcass was fabricated in the following subprimals: rack, roast ready, frenched PSO 3x1" (IMPS 204C); shoulder, square-cut, boneless (IMPS 208); Denver ribs, skirt-off (IMPS 209A); Foreshank (IMPS 210); loin, short-cut, trimmed PSO 0x0" (IMPS 232A); flank untrimmed (IMPS 232E); leg, hindshank (IMPS 233F); and leg, shank-off, boneless (IMPS 234A). Lastly, all lambs were utilized to determine dry matter, moisture, crude protein, crude fat, ash, vitamins A and E, trace minerals, and fatty acids. No interactions were found between treatment and gender for any characteristic, so treatment and gender were analyzed separately. Lambs of ewes that were nutritionally restricted were smaller in size with less fat. Lambs of the realimented group had more fat than either the control or the half ration groups. Rams had more percent lean content than ewes, which was to be expected. Results of this study provide insight on the effect of nutrient restriction on lamb growth and development, as well as nutrient content of American lamb.Item Open Access The impact of shear rate and reverse flow on cardiac morphogenesis and gene expression in the embryonic zebrafish heart(Colorado State University. Libraries, 2015) Zeller, Molly J., author; Garrity, Deborah M., advisor; Mykles, Donald, committee member; Bedinger, Patricia, committee member; Dasi, Lakshmi Prasad, committee memberMissteps in formation of the embryonic heart can have drastic consequences, making cardiac malformations a common human birth defect. During development, biomechanical factors including shear stress and reverse flow impact cardiogenesis. Shear stress is an epigenetic biomechanical force acting upon endothelial cells. Normally, a short period of reverse flow occurs prior to atrioventricular valve formation during ventricle systole and atrial diastole. The goal of our research is to investigate how altered biomechanical forces acting on endocardial cells lead to genetic responses by the heart. The mammalian zinc finger transcription factor Krüppel-like factor 2 (KLF2) responds to shear stress signals. Here, we explore the zebrafish KLF genes: klf2a, klf2b, and klf4. Whole embryo RT-PCR indicates that the three genes are expressed throughout early development, with cardiac expression in all genes present by 48 hours post fertilization. To evaluate how changes in biomechanical environments trigger altered gene expression in endocardial cells, we used comparative qPCR to quantify klf2a, klf2b, and klf4 expression levels in embryonic hearts with altered shear stress or reverse flow. Knockdown of the hematopoiesis gene gata2 was found to decrease blood viscosity, thereby decreasing both shear stress and reverse flow. Knockdown of contractility gene filaminCb was found to decrease shear stress but significantly increase reverse flow. Using high-speed imaging we quantified these forces and correlated changes in klf2a, klf2b, and klf4 expression. klf2a expression levels decreased in response to changes in both blood viscosity and cardiac contractility. klf2b and klf4 expression levels did not significantly change with these changes in biomechanical stresses. Our investigations considered the impact of blood viscosity versus cardiac contractility on KLF expression and determined that klf2a is a flow response gene. This data confirms previous studies that klf2a is in fact a flow response gene and shows that klf2b and klf4 are not responsive to changes in blood viscosity or cardiac contractility. Future studies will use transcriptomic approaches to identify genes regulated by the KLF family in response to shear stress and reverse flow cues.Item Open Access The voltage gated calcium channel β2 protein is required in the heart for control of cell proliferation and heart tube integrity(Colorado State University. Libraries, 2011) Chernyavskaya, Yelena, author; Garrity, Deborah Marie, advisor; Mykles, Donald, committee member; Miller, Charles, committee member; Reddy, A. S. N., committee memberL-type calcium channels regulate calcium (LTCC) entry into cardiomyocytes. CACNB2 (β2) LTCC auxiliary subunits traffic the pore-forming CACNA subunit to the membrane and modulate channel kinetics. β2 is a Membrane Associated Guanylate Kinase (MAGUK) protein. A major role of MAGUK proteins is to scaffold cellular junctions and multi-protein complexes. To investigate developmental functions for β2.1, we depleted it in zebrafish using morpholinos. β2.1-depleted embryos developed cardiac edema and lethal cardiac defects. Ventricular cardiomyocytes proliferated at a slower rate, and failed to elongate their cell shape, which led to dysmorphic cardiac morphology and weakened contractility. Reduction in proliferation was marked by smaller heart fields and an increase in bmp4, an anti-proliferative marker. Thus, β2.1 helps regulate heart size by regulating the rate of mitosis and bmp4 expression in the ventricle. Additionally, cardiomyocytes depleted for β2.1 failed to accumulate N-cadherin at the membrane, and dissociated easily from neighboring myocytes under stress. Hence, we propose that β2 could function as a MAGUK scaffolding unit to maintain N-cadherin-based adherens junctions and heart tube integrity. To test this hypothesis we mutated the β2.1 residues necessary for interaction with the LTCC and observed its expression in cardiomyocytes using a GFP tag. Mutant β2.1 was still able to localize to the membrane supporting the possibility that it has a role in maintaining other protein complexes, such as adherens junctions.Item Open Access Tissue-specific seasonal changes in mitochondrial respiratory function and membrane composition in the golden-mantled ground squirrel(Colorado State University. Libraries, 2016) Heim, Ashley, author; Florant, Gregory, advisor; Chicco, Adam, committee member; Mykles, Donald, committee memberMammals that hibernate, such as the golden-mantled ground squirrel (Callospermophilus lateralis; GMGS), cease to feed, reduce metabolic rate, and lower body temperature (Tb) during the winter months, surviving almost exclusively on the oxidation of lipids from endogenous fat stores. Whether mitochondria, the cellular sites of oxidative metabolism, undergo changes in response to low Tb, hypometabolism, and decreasing ambient temperature (Ta) to facilitate this remarkable phenotype is unclear. It has been postulated that changes in mitochondrial membrane composition reported in response to cold exposure in some species may facilitate maintenance of respiratory enzyme function with decreasing Tb. However, no studies to date have investigated the function and membrane fatty acid (FA) composition of mitochondria from different tissues across seasons in a hibernating mammal. We hypothesize that tissue-specific differences in mitochondrial respiration occur across seasons in the GMGS that may parallel distinct changes in mitochondrial membrane composition. We compared the respiration, substrate preference, and membrane composition of GMGS mitochondria isolated from liver, heart, skeletal muscle, and brown adipose tissue (BAT) from summer, fall (prehibernating), winter (hibernating), and spring (posthibernating) seasons. Maximal mitochondrial oxidative phosphorylation (OXPHOS)-supported respiration was determined by high-resolution respirometry at 37ᵒC in the presence of saturating concentrations of ADP and respiratory substrates. Mitochondrial membrane FA composition was determined by gas chromatography on phospholipid fractions obtained from isolated mitochondria. Maximal OXPHOS assayed at 37ᵒC tended to increase from summer to winter in liver, heart and BAT, and decrease in skeletal muscle, with tissue- and season-specific changes in pyruvate versus FA (palmitoylcarnitine) oxidation capacity. OXPHOS capacity was uniformly suppressed by decreasing Ta in all tissue mitochondria from torpid GMGS in the winter, despite widely variable changes in mitochondrial membrane composition across tissues and seasons. Taken together, these findings argue against a consistent relationship between changes in mitochondrial membrane composition and respiratory function across seasons in GMGS, but highlight distinct tissue- and season-specific differences that may have important biological effects that remain to be elucidated.Item Open Access Tugging on the heartstrings: determining the function of Tbx5 in early cardiac development(Colorado State University. Libraries, 2012) Parrie, Lindsay E., author; Garrity, Deborah, advisor; Bouma, Gerrit, committee member; Mykles, Donald, committee member; Reddy, A. S. N., committee memberDuring cardiac morphogenesis, the vertebrate heart acquires a characteristic three dimensional shape well-suited for efficient function. The morphology of the developing cardiac organ reflects a series of changes in the cardiomyocytes themselves, which must become specified, migrate, proliferate, grow in size, alter their shape and adhesive properties, and develop ultrastructure, among other differentiated characteristics. Mutation of the T-box transcription factor tbx5 leads to embryonic lethal cardiac phenotypes and forelimb malformations in vertebrate models. Haploinsufficiency of Tbx5 results in Holt-Oram Syndrome (HOS), a human congenital disease characterized by cardiac and forelimb defects. Homozygous mutation of zebrafish tbx5a in heartstrings (hst) embryos also leads to lethal defects in cardiac looping morphogenesis and prevents initiation of pectoral fin formation. Here I describe a new hst mutant allele (tbx5as296) which encodes a premature stop codon within the tbx5a T-box region, a location likely to generate a full loss-of-function allele. Data from comparative genetics and immunoblot analyses indicate that both alleles are null. I find that mutants completely lacking Tbx5a generated normal cardiomyocyte numbers in early chamber morphogenesis stages. Moreover, in situ hybridization data and functional assays support the idea that venous differentiation is not seriously impaired in zebrafish mutants, in contrast to mouse. However, cardiac cell size was significantly smaller in both chambers of tbx5a mutants. Hearts stalled early in the process of cardiac looping, but cell shape changes associated with chamber ballooning surprisingly still occurred. These studies point to a critical role for Tbx5a in growth-related aspects of cardiac differentiation, and suggest that morphologic events of cardiac looping morphogenesis and chamber ballooning are genetically separable. A second zebrafish tbx5 paralog was recently described, termed tbx5b, which showed a lower amount of sequence conservation than is typical for a T-box gene. Based on overlapping expression patterns within the embryonic heart, I hypothesized that functional redundancy between tbx5a and tbx5b might reduce the severity of cardiac phenotypes for tbx5a mutant embryos. I here report that the cardiac phenotypes in tbx5b-depleted fish were similar, but not identical, to those of homozygous tbx5a mutants. In addition, tbx5b-depletion led to defects in the timing and morphogenesis of pectoral fin outgrowth. Somewhat surprisingly, simultaneous depletion of both Tbx5 gene paralogs did not lead to more severe cardiac phenotypes, and injection of wild-type mRNA was not sufficient to cross-rescue the phenotypes of the paralogous gene. In the heart, tbx5a and tbx5b appear to have related essential functions that are nevertheless independently required. In the fin, tbx5a alone was required for fin bud initiation, but both genes are independently required for patterning and morphogenesis. Therefore, this work demonstrates a functional divergence between the two zebrafish tbx5 paralogs.