No description available.

<p>Dhurrin is a cyanogenic glucoside (CG), an important compound that can interplay with primary and secondary metabolism in sorghum. Dhurrin metabolism contributes to insect resistance, growth, nitrogen (N) metabolism, drought tolerance, and safety for animal consumption when used as a forage. Through chemical mutagenesis with ethyl methanesulfonate (EMS), a mutation in the gene encoding CYP79A1 (<i>cyp79a1</i>), the first enzyme in the biosynthetic pathway of dhurrin, was discovered that inhibits the production of dhurrin. The acyanogenic phenotype of this mutant could be a major benefit in reducing the risk of hydrogen cyanide (HCN) toxicity within animals; however, understanding the effects of inhibiting dhurrin biosynthesis is important in understanding metabolic tradeoffs that could occur. This dissertation describes research to assess impacts and tradeoffs of the dhurrin-free trait on susceptibility to Fall Armyworm [<i>Spodoptera frugiperda</i> (J.E. Smith)] (FAW) feeding, seedling growth, effects on post-flowering drought tolerance, cold stress and utilization as a forage. Insect susceptibility and seedling growth were examined using near-isogenic lines (NILs) within the greenhouse utilizing non-destructive phenotyping technologies for green plant area and in the field comparing total leaf area and dry weight. Post-flowering drought stress was induced within a greenhouse, growth chamber, and field environments. The <i>cyp79a1 </i>mutation was tested in NILs, a near-isogenic backcross (NIBC) population, and near-isogenic hybrids (NIH), to understand the impacts of the <i>cyp79a1 </i>mutation<i> </i>on the stay-green trait. Palatability as forage was examined by comparing the feeding preference of ruminant animals with multiple conventional hybrids and an experimental hybrid carrying the <i>cyp79a1</i> mutation. This preference was also examined using a set of NILs varying in the <i>cyp79a1 </i>mutation<i>. </i> Safety was assessed in preference trials by testing for HCN release before grazing. To further our understanding of the benefits of sorghum as a forage, the dhurrin-free experimental hybrid was compared to seven conventional hybrids as a dry product. The dry sorghum product was tested for the release of HCN and dhurrin content. Lastly, the effects of low temperatures and frost were assessed for their effects on the production of dhurrin in cyanogenic and dhurrin-free sorghum genotypes. </p> <p>Overall, the biosynthesis of dhurrin had a significant effect on the deterrence of FAW and on the growth of sorghum seedlings. Dhurrin-free lines were more susceptible to FAW feeding but also exhibited a significantly higher growth rate. Dhurrin-free lines and hybrids only exhibited a slight increase in susceptibility to post-flowering drought stresses with only one dhurrin-free hybrid discovered to senesce faster than its wild-type NIH. Comparisons of the effects of dhurrin biosynthesis on stay-green in a NIBC population in Tx642 (B35), one of the most important sources of the stay-green trait, did not show any variation in chlorophyll concentration (CC) and normalized difference vegetation index (NDVI). Analyses of the impact of dhurrin on palatability as a forage showed that ewes preferred grazing on the dhurrin-free hybrids and NILs, showing that the ewes were able to detect the presence or absence of dhurrin while feeding. Experiments to assess the safety and stability of dhurrin in dried plant material demonstrated that dhurrin content did not change during drying and HCN was released after rehydration. Furthermore, high levels of HCN were immediately released when rumen fluid was added to dried plant materials containing dhurrin; however, no detectable HCN was released from dhurrin-free genotypes. Finally, sorghum plants exposed to freezing temperatures exhibited an increase in dhurrin content in conventional sorghum hybrids while no detectable dhurrin was noted within <i>cyp79a1 </i>mutants. </p> <p>Taken together, these studies demonstrate pleiotropic effects for the <i>cyp79a1 </i>mutation. Dhurrin-free genotypes were more susceptible to insect herbivory and may be slightly more susceptible to post-flowering drought within the hybrids; however, these genotypes exhibited higher seedling growth rates, feeding preference by ewes, no release of HCN in fresh or dry plant material, and frost did not cause an increase in dhurrin content.</p>

cAMP receptors contain highly conserved cAMP binding pockets, in part responsible for allosteric activation, yet CBDs exhibit a wide array of cAMP binding affinities. While several cAMP:CBD crystallographic structures have been solved, they are insufficient to explain differences in cAMP:CBD affinities. We hypothesize that it is the position of the apo autoinhibitory equilibrium and/or a change in the state-specific association constants of the active and inactive CBD forms that are primarily responsible for modulating ~1000-fold difference in cAMP affinities. Interestingly, we discovered that PKARIα and HCN2 have comparable state-specific association constants, suggesting that the position of the apo autoinhibitory equilibrium is primarily responsible for the large difference in observed cAMP affinities in these systems. In addition, the individual components of the cAMP binding pocket (i.e. BBR, PBC, and lid) show functional variability across different CBDs. In RIα, both the BBR and lid are dispensable for high affinity cAMP binding, leaving the PBC as the key determinant of cAMP affinity. Interestingly, in addition the PBC:cAMP contact side-chains, non-contact side-chains are also important in modulating cAMP affinity (ie. L201 and Y205). Further dissection of the contributions arising from the apo pre-equilibrium and the cAMP binding pockets is required to better understand cAMP affinity and selectivity. / Thesis / Master of Science (MSc)

No description available.

1/ Évaluation de l'activité anti-proliférative de la canthin-6-oneLa canthin-6-one est un alcaloïde d'origine végétale dont les propriétés antipyrétique et antiparasitaire sont utilisées en médecine traditionnelle. Une meilleure connaissance de ses propriétés anti-prolifératives et de son mode d'action sont nécessaires. Nous avons observé une inhibition complète de la prolifération de lignées cancéreuses en présence de canthin-6-one ayant pour origine une accumulation des cellules dans la phase G2/M du cycle cellulaire. La conservation phylogénétique des mécanismes du cycle cellulaire nous a permis d'utiliser la levure Saccharomyces cerevisiae pour rechercher des gènes cibles de la canthin-6-one. Nous avons identifié deux gènes de résistance correspondants à une pompe d'efflux et à une enzyme impliquée dans le contrôle qualité de la réplication à la transition G2-M.2/ Rôle de la signalisation calcique dans la différenciation des photorécepteursUne meilleure connaissance des mécanismes de différenciation des photorécepteurs est importante pour la lutte contre les dégénérescences rétiniennes. Un délai d'une semaine, entre la détermination des précurseurs de photorécepteurs et l'activation transcriptionnelle de gènes de fonction, suggère l'attente d'un signal important pour cette étape de la différenciation. Par une approche pharmacologique in vitro, nous avons pu montrer que l'activation du gène de la rhodopsine dépend d'une voie de signalisation impliquant un canal activé en hyperpolarisation (HCN), des canaux calciques de type T ou R et de l'activité de la kinase calmoduline-dépendante. Le profil d'expression du canal HCN1 dans la rétine embryonnaire suggère qu'il pourrait jouer un rôle limitant dans ce mécanisme. / 1/ Evaluation of canthin-6-one anti-proliferative properties Canthin-6-one is an alkaloid molecule produced by tropical plants used in traditional medicine for its antipyretic and antiparasitic properties. Evidence-based medicine requires better knowledge of canthin-6-one's anti-proliferative effects and mode of action. In the presence of canthin-6-one, we demonstrated a complete growth inhibition of human cancer cells due to the accumulation of cells in the G2/M phases of the cell cycle. Cell cycle pathways being evolutionarily conserved in eukaryotes, we used the yeast Saccharomyces cerevisiae as a model system to further analyze the mode of action of canthin-6-one. A yeast genomic library was screened for suppressors of canthin-6-one toxicity and two resistance genes were identified. One encodes a transporter, probably involved in canthin-6-one efflux. The other one encodes an enzyme involved in DNA quality control.2/ Role of calcium signaling in photoreceptor differentiationThe design of new treatments for retinal degenerations should benefit from better knowledge of photoreceptor differentiation. During retinal differentiation in chicken embryos there is a long time lag between commitment to the photoreceptor fate and transcriptional activation of rhodopsin gene, suggesting committed precursors are in a standby state waiting for a signal to activate opsin gene expression. We demonstrate by a pharmacological approach in vitro that rhodopsin gene activation depends on a signaling pathway involving hyperpolarisation-activated channels (HCN), T- or R-type calcium channels and calmodulin-dependent protein kinase. The expression profile of HCN1 in the embryonic retina suggests it may be a limiting factor for rhodopsin gene activation.

Les canaux nucléotidiques cycliques activés par hyperpolarization (HCN) sont un type de canaux ioniques voltage-dépendants qui contrôlent l'activité rythmique et la plasticité synaptique dans le cœur et le cerveau. Ces canaux permettent aux ions K+ et Na+ de passer, créant ainsi un courant entrant lors de l'hyperpolarization de la membrane. En raison de ses propriétés biophysiques inhabituelles, ce courant est appelé courant «If» ou courant d'hyperpolarization «Ih». Des anomalies du courant Ih sont associées à des arythmies et des troubles neurologiques, y compris l'épilepsie. On constate que différentes molécules modulent ce courant. Des résultats expérimentaux ont montré que les lipides jouent un rôle dans le déplacement de la dépendance en tension des canaux HCN vers des tensions plus positives ou dépolarisées. Le phosphatidylinositol 4,5-bisphosphate de phospholipide endogène et exogène, ou PI (4,5) P2, régule les canaux HCN en déplaçant l'ouverture du canal vers une tension plus dépolarisée. Cette modulation est supposée être par interaction directe de PI (4,5) P2 avec le canal HCN. Ici, nous utilisons la dynamique moléculaire et l'amarrage pour explorer et identifier le site de liaison grâce à l'analyse des contacts et de la stabilité des liaisons hydrogène impliquées dans les molécules de phsiphoinositide et l'interaction des canaux HCN. Nous proposons LYS et ARG du domaine HCN et S3 pour être des résidus clés dans le site de liaison à travers lequel les molécules de phosphoinositide peuvent potentiellement activer le canal. / Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a type of voltage gated ion channels that control rhythmic activity and synaptic plasticity in the heart and brain. These channels allow K+ and Na+ ions to pass, thereby creating an inward current upon hyperpolarization of the membrane. Due to its unusual biophysical properties, this current is called funny « If» or hyperpolarization « Ih » current. Abnormalities in Ih current are associated with arrythmia and neurological disorders including epilepsy. Different molecules are found to modulate this current. Experimental results have shown that lipids play a role in shifting the voltage dependence of HCN channels to more positive, or depolarized voltages. Both endogenous and exogenous phospholipid phosphatidylinositol 4,5-bisphosphate, or PI(4,5)P2, regulates HCN channels by shifting the opening of the channel to a more depolarized voltage. This modulation is postulated to be through direct interaction of PI(4,5)P2 with the HCN channel. Here, we use molecular dynamics and docking to explore and identify the binding site through analysis of the contacts and stability of the hydrogen bonds involved in phosphoinositide molecules and HCN channel interaction. We propose LYS and ARG residues of the HCN domain and S3 to be key residues in the binding site through which phosphoinositide molecules can potentially activate the channel.

Die vorliegende Studie untersuchte die Wiederholbarkeit der Aufzeichnung von Unterkiefer-bewegungen und die Steilheit der Kiefergelenkbahn in Abhängigkeit von der Befestigungs-methode der Registrierapparatur an der Unterkieferzahnreihe. Der Untersucher zeichnete dazu in zwei zeitlich getrennten Sitzungen Protrusions- und Mediotrusionsbewegungen von Probanden auf. Er befestigte dabei den Registrierbogen mit drei unterschiedlichen Verfah-ren am Unterkiefer: mit einem okklusionsbedeckenden, periokklusalen Löffel, mit einer Okklusionsklemme und einem paraokklusalen Löffel. Zwei Methoden bedecken die Unterkie-ferzahnreihe und erhöhen die vertikale Kieferrelation. Bei der dritten Methode hingegen befestigte der Untersucher den Unterkieferbogen an den Labialflächen der Unterkieferzähne mit ungestörter habitueller Interkuspidation. Die mittlere, über alle Befestigungsmethoden gemessene, sagittale Gelenkbahnneigung be-trug 44,74 ° bei 3 mm und 42,65 ° bei 5 mm Gelenkbahn. Der Bennett-Winkel nahm Werte von 10,96 ° (3 mm) und 10,17 ° (5 mm) an. Die sagittale Kiefergelenkbahn differierte bei den unterschiedlichen Befestigungsmethoden statistisch signifikant und klinisch relevant. Die Messung mit paraokklusaler Befestigung des Unterkieferbogens erzielte sagittale Kondylen-bahnneigungen, die im Mittel 7,94 ° (3 mm) und 9,46 ° (5 mm) steiler waren als die mit der Okklusionsklemme gemessenen Neigungen. Die Wiederholbarkeit war bei allen Gruppen innerhalb einer Sitzung exzellent, unterschied sich jedoch beim Vergleich beider Sitzungen statistisch signifikant. Die Gelenkbahnaufzeichnung mittels paraokklusaler Befestigung des Unterkieferbogens war exzellent wiederholbar. Unter Benutzung des periokklusalen Löffels und der Okklusionsklemme sank die Reliabilität statistisch signifikant und klinisch relevant ab. Die Untersuchung zeigte darüber hinaus, dass verschiedene Messsysteme mit unterschiedli-chen Bezugsebenen und -punkten arbeiten und die in der Literatur zu findenden Werte für Gelenkbahnneigungen daher kaum vergleichbar sind. Für die Programmierung virtueller und voll adjustierbarer Artikulatoren sollte daher, wenn möglich, die paraokklusale Befestigung für die Messung der Kiefergelenkbahnen Anwendung finden.

Neurons perform complex computations, communications and precise transmissions of information in the form of action potentials (APs). The high level of heterogeneity and complexity at all levels of organization within a neuron and the functional requirement of highly permeable cell membranes leave neurons exposed to damage when energy levels are insufficient for the active maintenance of ionic gradients. When energy is limiting the ionic gradient across a neuron’s cell membrane risks being dissipated which can have dire consequences. Other researchers have advocated “generalized channel arrest” and/or “spike arrest” as a means of reducing the neuronal permeability allowing neurons to adjust the demands placed on their electrogenic pumps to lower levels of energy supply. I investigated the consequences of hypoxia on the propagation of a train of APs down the length of a fast conducting axon capable of transmitting APs at very high frequencies. Under normoxic conditions I found that APs show conduction velocities and instantaneous frequencies nearly double that of neurons experiencing energy limiting hypoxic conditions. I show that hypoxia affects AP conduction differently for different lengths of axon and for APs of different instantaneous frequencies. Action potentials of high instantaneous frequency in branching lengths of axon within ganglia were delayed more significantly than those in non-branching lengths contained within the connective and fail preferentially in branching axon. I found that octopamine attenuates the effects of hypoxia on AP propagation for the branching length of axon but has no effect on the non-branching length of axon. Additionally, for energetically stable cells, application of the anti-diabetic medication metformin or the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 resulted in a reduced performance similar to that seen in neurons experiencing energetic stress. Furthermore both metformin and ZD7288 affect the shape of individual APs within an AP train as well as the original temporal sequence of the AP train, which encodes behaviourally relevant information. I propose that the reduced performance observed in an energetically compromised cell represents an adaptive mechanism employed by neurons in order to maintain the integrity of their highly heterogeneous and complex organization during periods of reduced energy supply. / Thesis (Master, Biology) -- Queen's University, 2011-10-07 14:41:46.972

SH3 and multiple ankyrin repeat domains 3 (SHANK3) is a multidomain scaffold protein that is highly augmented in the postsynaptic density (PSD) of excitatory glutamatergic synapses within the central and peripheral nervous systems. SHANK3 links neurotransmitter receptors, ion channels, and other critical membrane proteins to intracellular cytoskeleton and signal transduction pathways. Mutations in SHANK3 are linked with a number neuropsychiatric disorders including autism spectrum disorders (ASDs). Intellectual disability, impaired memory and learning, and epilepsy are some of the deficits commonly associated with ASDs that result from mutations in SHANK3. Interestingly, these symptoms show some clinical overlap with presentations of human neurological disorders involving hyperpolarization-activated cyclin nucleotide-gated (HCN) channels. In fact, it has recently been demonstrated in human neurons that SHANK3 haploinsufficiency causes Ih-channel dysfunction, and that SHANK3 has a physical interaction with HCN channels via its ANKYRIN repeat domain. These insights suggest that SHANK3 may play important roles in HCN channel expression and function, and put forward the idea that HCN channelopathies may actually encourage some of the symptoms observed in patients with SHANK-deficiency related ASDs. In this study, we provide preliminary data that suggests the ANK domain of SHANK3 interacts with COOH portion of HCN1. We also exploited the differences between two mouse models of autism to show that a subset of SHANK3 isoforms may be involved in the proper expression and function of HCN channels. We found that HCN2 expression is significantly decreased in a mouse model lacking all major isoforms of SHANK3 (exons 13-16 deleted; Δ13-16), while HCN2 expression is unaltered in a mouse model only lacking SHANK3a and SHANK3b (exons 4-9 deleted; Δ4-9). Surprisingly, we also found that HCN4 expression is altered in SHANK3Δ13-16, but not SHANK3Δ4-9. Taken together, our results show HCN channelopathy as a major downstream carrier of SHANK3 deficiency.

Trigeminal neuropathic pain (TNP) is a condition that occurs when one or more branches of the trigeminal nerve are insulted. Trigeminal neuropathic pain has been shown to be refractory to treatment. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate neuronal excitability in both the peripheral and central nerve systems. Emerging evidence indicates that HCN channels are involved in the development and maintenance of chronic pain, however, the impact of thalamic HCN channel activity on TNP has yet to be elucidated. In this report, we used a chronic constriction of the distal infraorbital nerve (dIoN-CCI) to induce TNP in rats. By infusing HCN channel blockers into the ventral posteromedial (VPM) nucleus of the thalamus in dIoN-CCI rats, we demonstrated that inhibition of HCN channel activity ameliorated TNP. We found that the HCN blocker ZD7288 and the clinical drug ivabradine dose-dependently attenuated both evoked and none-evoked nociceptive behaviors in dIoN-CCI rats. Electrophysiological measurements showed the expression of HCN current (Ih) in the thalamocortical neurons in the VPM was sensitive to the HCN channel modulator cyclic adenosine monophosphate (cAMP), suggesting a contribution of the HCN2 subunit in thalamic HCN current. In the thalamus, surface expression of the HCN2 subunit was increased in dIoN-CCI rats. Taken together, we propose that an increase in HCN channel activity in the thalamus in the ascending nociceptive pathway contributed to trigeminal neuropathic pain.