Browsing by Author "Kanatous, Shane, committee member"

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Open Access
Characterization of cyclic nucleotide phosphodiesterases in the transcriptome of the crustacean molting gland
(Colorado State University. Libraries, 2019) Rifai, Nada Mukhtar, author; Mykles, Donald L., advisor; Garrity, Deborah, committee member; Kanatous, Shane, committee member; Di Pietro, Santiago, committee member
Molting in crustaceans is a complex physiological process that has to occur in order for the animal to grow. The old exoskeleton must be discarded and a new one to be formed from the inside out. Molting is coordinated and regulated mainly by two hormones; steroid hormones named ecdysteroids, which are synthesized and secreted from a pair of Y- organs (YOs) that are located in the cephalothorax and a neuropeptide hormone, the molt inhibiting hormone (MIH), which is secreted from the X-organ/sinus gland complex located in the eyestalks. Molting is induced when MIH is decreased in the blood (hemolymph) which in turn stimulates the YOs to produce and secrete ecdysteroids (molting hormones). There are four distinctive physiological states that the YO can be in throughout the molt cycle; the transition of the YO from the "basal" to the "activated" state happens when the animal enters premolt. During mid-premolt, the YO transitions to the "committed" state, in which the YO becomes insensitive to MIH. In this state, the circulating hemolymph contains high levels of ecdysteroids, which increase to a peak before the actual molt (ecdysis) happens. The YO transitions from the committed to the repressed state in late premolt. Finally, the YO returns back to the basal state in the postmolt stage. MIH binds to membrane receptors, activating a signal transduction pathway divided into "triggering" and "summation" phases. A transient increase in cAMP during the triggering phase leads to prolonged cGMP-dependent suppression of ecdysteroidogenesis during the summation phase. This allows for sustained inhibition of the YO between MIH pulses in the intermolt animal. Cyclic nucleotide phosphodiesterases (PDEs) play an important role by controlling cAMP and cGMP levels. PDEs hydrolyze the phosphodiester bond in cAMP and cGMP to AMP and GMP, respectively. Mammals have 21 PDE genes that are categorized into 11 families, designated PDE1 to PDE11. Each PDE family has specific catalytic and biochemical properties and tissue distributions. Eight contigs encoding full-length PDE sequences were identified in the G. lateralis Y-organ transcriptome. Seven contigs encoding four full-length PDE sequences and three contigs encoding partial-length PDE were identified in the Carcinus maenas transcriptome. Multiple sequence alignments showed high sequence identities with orthologs from other species in catalytic (PDEase) and other conserved functional domains. Sequence analysis assigned the Gl-PDE sequences and Cm-PDE sequences to PDE1, PDE2, PDE3, PDE4, PDE5, PDE7, PDE8, PDE9, and PDE11 classes, indicating a high diversity of PDE genes in decapod crustaceans. The reduced sensitivity to MIH by the committed YO is associated with a large increase in PDE activity, which suggests that PDEs modulate the response to neuropeptide during the molt cycle. Non-hydrolyzable analogs of cAMP and cGMP inhibit YO ecdysteroid secretion in-vitro. Moreover, C. maenas YO ecdysteroidogenesis is inhibited by IBMX, a general PDE inhibitor, and Zaprinast, a specific PDE5 inhibitor. Rolipram, a specific PDE4 inhibitor, has no effect. These data suggest that PDE5 activity modulates the effect of MIH on YO ecdysteroidogenesis. RNA-seq data from MLA showed different mRNA levels for the different PDEs; PDE1 and PDE2 showed a similar pattern as they both increased in intermolt (IM) then decreased dramatically in early premolt (EP), mid premolt (MP), late premolt (LP), and post molt (PM). PDE4 increased in IM followed by a slight decrease and increase in EP and MP then a sharp decline in both LP and PM. Both PDE5 and PDE9 were similar in terms they increased in IM followed by a sharp decrease in EP, MP, LP and they differed as PDE5 increased slightly in PM whereas PDE9 remained decreased. PDE7 began with an increase in IM then a decline with a constant expression level in both EP and MP followed by dramatic decline in LP and PM. PDE11 showed a typical pattern consistent with the ecdysteroid expression level as it began with a slight increase in IM followed by an increased in EP and reached a peak in MP then declined in a dramatic way in LP and continued decreasing in PM. Taken together, the data suggest that PDE5 and PDE11 play a role in regulating cyclic nucleotide levels in the YO.
Open Access
Development of an asymmetric intermolecular stetter reaction and organocatalyst design
(Colorado State University. Libraries, 2012) Glover, Garrett S., author; Rovis, Tomislav, advisor; Wood, John L., committee member; Kanatous, Shane, committee member
The intermolecular Stetter represents a powerful carbon-carbon bond forming reaction that involves addition of an acyl anion equivalent to an untethered Michael acceptor. Despite the appeal of rendering the intermolecular reaction asymmetric, this area remains particularly underdeveloped. We describe a catalytic asymmetric intermolecular Stetter reaction for the coupling of 2-pyridinecarboxaldehydes and β-substituted vinyl triflones. In pursuit of increasing the efficiency of this reaction, we developed a series of base-activated NHC pre-catalysts that are capable of catalyzing the intramolecular Stetter reaction without an external base. As an indirect result of our attempts to form activated pre-catalysts, we discovered a catalyst hydration product, that itself generates free-carbene in solution. Additionally, we gathered conclusive evidence for a previously unknown SNAR type decomposition pathway of an electron deficient triazolinylidene catalyst.
Open Access
Influence of exercise modality and modality-specific training on endurance exercise performance in hypoxia
(Colorado State University. Libraries, 2017) Theisen, Jeremy Kenneth, author; Bell, Christopher, advisor; Dinenno, Frank, committee member; Kanatous, Shane, committee member
INTRODUCTION: In hypoxia, endurance exercise performance is impaired. The magnitude of impairment may be variable between individuals and exercise modalities. The purpose of this study was to determine the influence of exercise modality and modality-specific training on hypoxia-mediated performance decrements. METHODS: In a randomized cross-over design, endurance trained cyclists (4 males, 3 females) and rowers (5 males, 3 females) performed exercise on both cycling and rowing ergometers. On separate occasions, participants completed graded exercise tests in normoxia (FiO2= 0.21), and standardized exercise (15 minutes, 100 W) and time trials (4 km cycling, 2 km rowing) in normoxia and hypoxia (FiO2= 0.15). RESULTS: Hypoxia-mediated performance decrements were not different between cyclists and rowers (17±1 vs. 18±1%, p=0.189), cycling and rowing (18±2 vs. 16±2%, p=0473), or any combination of training or test modality (p=0.138). In rowers, peripheral oxygen saturation (SpO2) was lower at the end of rowing compared to cycling time trials (78±1 vs. 83±1%, p=0.002), and lower than that of cyclists at the end of rowing time trials (78±1 vs. 83±1%, p<0.001). DISCUSSION: Hypoxia-mediated performance decrements were not different between training modalities, test modalities, or any combination of the factors. We speculate that reduced SpO2 in rowers at the end of rowing time trials may be related to a greater active muscle mass, causing a rightward shift in the oxyhemoglobin dissociation curve and reduced transit time of blood in pulmonary capillaries. In conclusion, SpO2 may be related to active muscle mass during exercise and could potentially modulate performance in hypoxia.
Open Access
Influence of pre-fire beetle kill on post-fire hairy woodpecker (Leuconotopicus villosus) abundance in the Southern Rocky Mountains, Colorado
(Colorado State University. Libraries, 2025) Jones, Lilly N., author; McTigue, Leah, advisor; Kanatous, Shane, committee member
Increasing wildfire frequency and severity in the western United States, combined with widespread bark beetle outbreaks, have significantly altered forest ecosystems in the Rocky Mountains. These interacting disturbances are reshaping post-fire habitats, particularly for cavity-nesting birds such as the Hairy Woodpecker (Leuconotopicus villosus). This study aimed to assess how proximity to beetle-killed forest stands influenced Hairy Woodpecker relative abundance following the 2020 Cameron Peak Fire in the Southern Rocky Mountains, Colorado. We deployed automated recording units (ARUs) at 19 sites within and around the burn perimeter during summer 2023. Recordings of bird calls were analyzed using acoustic identification software to quantify woodpecker detection. Distances to beetle-killed areas were calculated using U.S. Forest Service data and geospatial analysis. A linear regression model revealed a significant negative relationship between woodpecker abundance and distance to beetle-killed forest stands (p < 0.01), with closer proximity associated with higher abundance. These results provide valuable insight into habitat selection by cavity-nesting woodpeckers, demonstrating how forest structure shaped by beetle activity can influence post-fire wildlife use. As climate change drives more frequent and intense disturbances, it is important that forest management strategies account for the lasting impacts of compound events like beetle outbreaks and wildfire to promote wildlife resilience.
Open Access
Progress toward the total synthesis of stemocurtisine and asymmetric synthesis of endoperoxide anticancer agents via Brønsted acid cascade catalysis
(Colorado State University. Libraries, 2012) Rubush, David Michael, author; Rovis, Tomislav, advisor; Kennan, Alan, committee member; Crans, Debbie, committee member; Chen, Eugene, committee member; Kanatous, Shane, committee member
A viable route toward the pyrido-azepine core of stemocurtisine involving an N-heterocyclic carbene catalyzed Stetter reaction has been realized. The key steps involve a formal [3+2] cycloaddition of enones with isocyanoacetates and a catalytic asymmetric intramolecular Stetter reaction. Additionally, a diastereoselective intramolecular Stetter reaction was achieved to access highly substituted pyrrolidines. Asymmetric Brønsted acid catalyzed cascade reactions were also investigated. A diastereoselective acetalization/oxa-Micahel cascade has been developed to provide dioxolanes and oxazolidines using diphenylphosphinic acid as a catalyst. The enantioselective variant of this reaction was explored with minor success. The desymmetrization of p-peroxyquinols using a Brønsted acid catalyzed acetalization/oxa-Michael cascade was achieved in high yields and selectivities for a variety of aliphatic and aryl aldehydes. Mechanistic studies suggest that the reaction proceeds through a dynamic kinetic resolution of the peroxy-hemiacetal intermediate. The resulting 1,2,4-trioxane products were derivatized and show potent cytotoxicity toward specific cancer cells.
Open Access
Regulation of Halloween genes and ecdysteroid responsive genes in molting gland of Blackback land crab Gecarcinus lateralis by molt-inhibiting hormone, mTOR and TGFβ signaling pathways pathways
(Colorado State University. Libraries, 2019) Benrabaa, Samiha Abd Al Salem, author; Mykles, Donald, advisor; Garrity, Deborah, committee member; Kanatous, Shane, committee member; Stargell, Laurie, committee member
Molting is necessary for growth and development in all arthropods. Halloween genes are expressed in the Y-organs (YO) and encodes cytochrome P450 enzymes. These enzymes catalyze the synthesis of ecdysteroid hormones that regulate the molt cycle. This hormone binds to ecdysteroid receptor to activate a cascade of ecdysteroid-responsive genes that effect tissue responses to hormone. We used P. leptodactylus Halloween gene and insect ecdysteroid-responsive gene sequences to extract and characterize the land crab orthologs in the YO transcriptome. This resulted in identification and characterization of eight ecdysteroidogenic genes that encode phantom, disembodied, spook, shadow, Cyp18a1, neverland, NADK and ALAS and nine ecdysteroid-responsive genes that encoded EcR, RXR, broad complex, E75, E74, Hormone receptor 4, Hormone receptor 3, forkhead box transcription factor (FOXO) and Fushi tarazu factor-1. Sequences were validated by end-point PCR and Sanger sequencing. We used phylogenetic analysis to infer evolutionary relationships among contig sequences and ortholog of Halloween genes and ecdysteroid-responsive genes in other species. The results showed the contig sequences clustered with their corresponding orthologous genes. Tissue distributions for spook and phantom showed significantly higher mRNA levels in the YO compared to other tissues. By contrast, the mRNA levels of NADK, ALAS, and all ecdysteroid-responsive genes were not higher in the YO than those in other tissues. These data show that the YO is the primary source of ecdysteroid production and that the YO can respond to ecdysteroid, suggesting a feedback regulation on ecdysteroid synthesis and secretion. qPCR was used to quantify gene expression of Halloween and ecdysteroid-responsive gene expression in the YO of Gecarcinus lateralis induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). ESA decreased mRNA levels of Gl-Phm, Gl-E75 and Gl-RXR at 3 days post-ESA. Gl-Dib and shadow increased at 3 days post ESA and decreased at 7 and 14 days post-ESA. Gl-Cyp18a1, Gl-BR-C, Gl-NADK and Gl-ALAS mRNA were higher at Day 0 and 1 post-ESA and lower at Day14 post ESA. Gl-HR3, Gl-HR4, and Gl-E74 were expressed at low levels. MLA lowered mRNA levels of Halloween genes, Gl-Nev, and Gl-E75, except Gl-dib, at premolt and postmolt stages. Gl-Dib, Gl-NADK, Gl-ALAS, and Gl-BR-C mRNA levels were not affected by molt stage. Gl-EcR, Gl-HR4 and Gl-HR3 mRNA levels were highest in premolt and lowest in postmolt. Gl- FOXO mRNA levels were highest in premolt and lowest in intermolt. These data suggest that molting has an indirect effect on the regulation of Halloween genes and that molting directly regulates HR3, HR4, RXR and FOXO to increase ecdysteroid synthetic capacity of the YO in premolt animals. The presence of EcR/RXR and ecdysteroid-responsive genes suggest that elevated ecdysteroid represses the YO at the end of premolt. TGFβ/activin signaling mediates the transition of the YO from the activated to the committed state, as SB431542 blocks this transition. G. lateralis were eyestalk-ablated to induce molting and injected with vehicle (DMSO) or SB431542 at Day 0. In controls, ESA increased hemolymph ecdysteroid titers at 3, 7 and 14 days post-ESA. There were significant increases in the mRNA levels of Gl-Nvd at 7 and 14 days post-ESA and other Halloween genes (Gl-Spo, Gl-Phm, Gl-Dib, and Gl-Sad), as well as Gl-CYP18a1, Gl-ALAS, Gl-NADK, Gl-BR-C,Gl- FOXO, Gl-EcR, and Gl-RXR, at 14 days post-ESA. SB431542 reduced hemolymph ecdysteroid titers at 7 and 14 days post-ESA compared to control animals, but titers were no different from controls at 1, 3, and 5 days post-ESA, indicating that SB431542 had no effect on YO activation. SB431542 blocked the increases in mRNA levels of Gl-Nvd, Gl-Spo, Gl-Phm, Gl-Dib, Gl-Sad, Gl-CYP18a1, Gl-ALAS, Gl-NADK, Gl-BR-C, Gl-EcR, and Gl-RXR by ESA. SB431542 had no effect on mRNA levels of the ecdysteroid-responsive genes Gl-HR3 Gl-HR4, Gl-E74, Gl-E75 and Gl-FTZ-F1. These data suggest that an Activin-like TGFβ factor stimulates YO ecdysteroidogenesis in the committed YO by up-regulating Halloween, Gl-BR-C, and Gl-FOXO genes.
Open Access
Role of stress-responsive genes in the molt cycle of decapod crustaceans
(Colorado State University. Libraries, 2019) López-Cerón, Diego Alejandro, author; Mykles, Donald, advisor; Garrity, Deborah, committee member; Kanatous, Shane, committee member; Di Pietro, Santiago, committee member
The main objective of this study was to understand the molecular mechanisms underlying the inhibition of molting by environmental stress in decapods. The general hypothesis describes that environmental stressors inhibit synthesis and secretion of molting hormones (ecdysteroids) by the molting gland, or Y-organ (YO), through stress response signaling genes in the mTOR signaling pathway. It is predicted that premolt animals of Gecarcinus lateralis will be more sensitive to thermal stress compared to those in intermolt stages, and that G. lateralis will be more sensitive to environmental stresses such as temperature or desiccation than Carcinus maenas. The aims of this study were to review of state of the art on molting in decapods, regulation of molt cycle by environmental stress, design of model of stress response in decapods (Chapter 1), Isolate, identify and characterize cDNAs encoding stress response genes in G. lateralis and C. maenas (Chapter 2), Quantify the effects of acute thermal stress on the Y-organ of Multiple Leg Autotomy (MLA) and Eyestalk Ablated (ESA) intermolt, premolt, and postmolt G. lateralis (Chapter 3), and Quantify the effects of desiccation on gene expression and mTOR activity in intermolt animals of C. maenas (Chapter 4).
Open Access
Site-selective functionalization of azines and polyazines via heterocyclic phosphonium salts
(Colorado State University. Libraries, 2020) Dolewski, Ryan D., author; McNally, Andrew, advisor; Paton, Robert, committee member; Henry, Chuck, committee member; Kanatous, Shane, committee member
Pyridine and diazines are frequently found in FDA approved drugs, biologically active compounds, agrochemicals, and materials. Given the importance of these structural motifs, direct methods that selectively functionalize pyridine and diazine scaffolds have been developed. These methods and their associated challenges are discussed in chapter one. In chapter two, a strategy to directly and selectively functionalize pyridines and diazines via heterocyclic phosphonium salts is presented. The process is broadly applicable for pyridines and diazines and the late-stage functionalization of pharmaceuticals. Four reaction manifolds are amenable to transforming heterocyclic phosphonium salts into valuable derivatives. In chapter three, inherent factors that control site-selectivity in polyazine systems are described along with mechanistically driven approaches for site-selective switching, where the phosphonium ion can be predictably installed at other positions in a polyazine system. The fourth chapter focuses on a new strategy to selectively alkylate pyridines via a traceless dearomatized phosphonium salt intermediate. Preliminary studies show this protocol is amenable to building-block pyridines, drug-like fragments and pharmaceuticals. A late-stage methylation strategy is also presented.
Open Access
Synthesis of nitrogen-containing molecules by zinc-catalyzed [4+2] cycloaddition and photoredox-catalyzed C-H functionalization
(Colorado State University. Libraries, 2017) Chu, John Chun Kit, author; Rovis, Tomislav, advisor; Kennan, Alan, committee member; Prieto, Amy, committee member; Kanatous, Shane, committee member
This work first describes an enantioselective Zn-catalyzed [4+2] cycloaddition of 1-azadienes and nitro-alkenes for the synthesis of medicinally valuable piperidines. The detrimental coordination of 1-azadienes to the Zn catalysts undermines the stereochemical control of the reaction. Fortunately, a novel bisoxazoline ligand limits this undesired coordination and delivers high stereoselectivity. Mechanistic studies suggest the reaction proceeds via a stepwise mechanism in which aza-Michael addition is followed by cyclization. This proposed mechanism also explains the successful cycloaddition between two electron-deficient reaction partners. Secondly, amide-directed carbon-carbon bond formation at unactivated sp3 C-H bonds has been achieved using photoredox catalysis. The reaction features a hydrogen atom abstraction from the C-H bond to a nitrogen radical generated from the amidyl N-H bond, leading to formation of a carbon-centered radical. Trapping of the resulting alkyl radical with an electrophilic alkene gives the desired C-C bond formation. Experimental evidence supports the generation of the nitrogen radical through a stepwise deprotonation/oxidation event in a closed catalytic cycle. The potential to incorporate other functionalities in the C-H bonds, as well as g functionalization of carbonyl compounds, is disclosed.
Open Access
The role of ecdysteroids on Myostatin and mTOR signaling gene expression in molt-dependent growth and atrophy of skeletal muscle in Gecarcinus lateralis and Carcinus maenas
(Colorado State University. Libraries, 2016) Cosenza, Kathy S., author; Mykles, Donald L., advisor; Quackenbush, Sandra L., committee member; Kanatous, Shane, committee member; DeLuca, Jennifer G., committee member
During premolt, increasing ecdysteroid levels are correlated with increased protein synthesis in the claw of decapod crustaceans. Increased protein synthesis is necessary to remodel the claw muscle in preparation for rapid hypertrophy immediately after ecdysis. Ecdysteroids are negatively correlated with G. lateralis-Myostatin (Gl-Mstn) mRNA levels, allowing upregulation of protein synthesis. Conversely, steroids upregulate Mstn expression in mammals. Glucocorticoids inhibit protein synthesis in mammals through downregulation of the mechanistic Target of Rapamycin (mTOR)-dependent protein synthesis pathway, and upregulation of Mstn. Here, we look at the relationship between ecdysteroids and the mRNA levels of Mstn and mTOR pathway components in different skeletal muscles of G. lateralis and C. maenas. The claw muscle and limb bud muscles respond to increasing premolt ecdysteroid levels with increased protein synthesis, while thoracic muscle does not change in protein synthesis. Our first hypothesis is that ecdysteroid levels will be negatively correlated with Mstn mRNA levels in the claw muscle and the limb bud muscles (muscles with increased protein synthesis), but not in thoracic muscle (no change in protein synthesis). Our second hypothesis is that Gl-Rheb mRNA levels of will be positively correlated with ecdysteroid levels, and negatively correlated with Mstn levels in the claw and limb bud muscles, but not in thoracic muscle. Our third hypothesis is that ecdysteroids directly regulate Gl-Mstn promoter expression through an ecdysone response element (EcRE) in the Gl-Mstn promoter. Through molt manipulations, or allowing natural molts, we showed that ecdysteroid levels were negatively correlated with Gl-Mstn mRNA levels, but not correlated with C. maenas-Mstn (Cm-Mstn) mRNA levels, in claw muscle. In limb bud muscle, there was no correlation between ecdysteroid levels and Gl-Mstn levels. Gl-Mstn levels remained very low, whether limb buds were growing or growth had been suspended. There were no correlations between ecdysteroids and Mstn mRNA levels in the thoracic muscle of either species. These data indicate that ecdysteroid regulation of Mstn is not consistent between species, and that ecdysteroid regulation is muscle specific. Contrary to our second hypothesis, Mstn was positively correlated with mTOR signaling components in the claw muscle of both G. lateralis and C. maenas, not negatively correlated. There was no correlation between Gl-Mstn and mTOR component mRNA levels in the limb buds. This indicates that in specific situations with dramatically changing protein synthesis, Mstn as a chalone that has a modulating effect, to prevent excessive protein synthesis. Using DNA walking, a putative EcRE was located in the promoter region of the Gl-Mstn gene. A heterologous ecdysteroid cell system was developed in mammalian cells to determine Gl-Mstn promoter activity in response to precisely controlled ecdysteroid levels. The heterologous system showed that the Gl-Mstn promoter was functional in this system, but we were unable to demonstrate direct regulation of the Gl-Mstn promoter by ecdysteroids. Further work with the heterologous cell system may determine whether the putative EcRE in the Gl-Mstn is functional.