Understanding the nature of adaptive evolution has been the recent focus of research detailing the genetic basis of adaptation and theoretical work describing the mechanics of adaptive evolution. Nevertheless, key questions regarding the process of adaptive evolution remain. Ultimately, a detailed description of the ecological context, evolutionary history, and genetic basis of adaptations is required to advance our understanding of adaptive evolution. To address some of the contemporary issues surrounding adaptive evolution, I examine phenotypic and genotypic changes in a snake feeding adaptation. Adaptations can arise through fixation of novel mutations or recruitment of existing variation. Some populations of the garter snakes Thamnophis sirtalis, T. couchii, and T. atratus possess elevated resistance to tetrodotoxin (TTX), the lethal toxin of their newt prey. I show that TTX resistance has evolved independently through amino acid changes at critical sites in a voltage-gated sodium channel protein (Nav1.4) targeted by TTX. Thus, adaptive evolution has occurred multiple times in garter snakes via de novo acquisition of beneficial mutations. Detailing the genetic basis of adaptive variation in natural populations is the first step towards understanding the tempo and mode of adaptive evolution. I evaluate the contribution of Nav1.4 alleles to TTX resistance in two garter snake species from central coastal California. Allelic variation in Nav1.4 explains 29% and 98% of the variation in TTX resistance in T. atratus and T. sirtalis, respectively, demonstrating that Nav1.4 is a major effect locus. The simple genetic architecture of TTX resistance in garter snakes may significantly impact the dynamics of trait change and coevolution. Patterns of convergent evolution are cited as some of the most compelling examples of the strength of natural selection in shaping organismal diversity. Yet repeated patterns may tell us as much about the constraints that restrict evolution as about the importance of natural selection. I present data on convergent molecular adaptations in parallel arms races between diverse snakes and amphibians from across the globe. Six snake species that prey on TTX bearing amphibians have independently acquired amino acid changes in Nav1.4. The derived mutations are clustered in two portions of the gene, often involving the same sites and substitutions. While a number of amino acid changes can make Nav1.4 insensitive to TTX, most of these negatively impact or abolish the ion-conducting function of the protein. Thus, intramolecular pleiotropy likely prevents most replacements from becoming fixed and imposes limits on protein evolution.

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No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-gated sodium channels (VGSCs) are dynamic membrane-spanning proteins crucial for determining the electrical excitability in nerve and muscle. VGSCs transition, or gate, between opened, closed, and inactivated states, in response to changes in transmembrane potential. Altered VGSC gating can affect electrical communication and is implicated in numerous channelopathies. Nav1.7, a VGSC isoform highly expressed in the peripheral nervous system, plays a unique role in pain perception as evidenced by single point missense mutations causing a spectrum of pain syndromes (inherited erythromelalgia; IEM and paroxysmal extreme pain disorder; PEPD) and nonsense mutations resulting in human insensitivity to pain (CIP). These studies indicate Nav1.7 is critical in pain transduction and, as such, structural perturbations to Nav1.7 affecting conformational stability and response to changes in transmembrane potential have the potential to cause pain. Therefore, the aims of this dissertation were to (1) examine the effects of PEPD mutations on the voltage-dependent properties Nav1.7; (2) investigate the effects Nav1.7 alternative splicing has on the impact of IEM and PEPD mutations; (3) evaluate the effects channelopathies, resulting from slowed inactivation, have on modulating an unusual type of sodium current that flows during membrane repolarization; and (4) determine the structural components involved in stabilizing Nav1.7 inactivation. Standard patch-clamp electrophysiology was used to study changes in channel properties. Results from this dissertation demonstrate that (1) PEPD mutations significantly shift the voltage-dependent properties of Nav1.7 channels, destabilize an inactivated state in a residue specific manner, and render nociceptive neurons hyperexcitable; (2) alternative splicing can functionally impact PEPD; (3) channelopathies, resulting from slowed inactivation in neuronal and muscle VGSC isoforms, increase an unusual sodium conductance that flows during repolarization; and (4) specific residues located in distinct regions of Nav1.7 serve as docking sites to stabilize inactivation at different membrane potentials. Overall, this dissertation answers key questions regarding the molecular mechanics required during inactivation and the biophysical consequences of Nav1.7 mutations implicated in painful disorders. The results of this dissertation are important for a more detailed understanding of pain perception and validate the applicability of studying Nav1.7 for discovery of therapeutic targets for treatment of pain. – Theodore R. Cummins, Chair

A well documented laboratory setup, leech preparation process, and bio-potential data recording process are needed. Repeatability and quality data recordings are essential and thus dictate the requirements of the laboratory setup and processes listed above. Advances in technology have both helped and hindered this development. While very precise equipment is required to record the low voltage bio-potentials, noisy electronic equipment and wires surrounding the work area provide high levels of interference. Proper laboratory setup and data recording processes, however, limit the unwanted interference. Quality data can only be recorded from a properly handled and prepared leech subject. Proper setup and procedures result in quality recordings which lend a clean signal for furthering the understanding of nerve functionality. The electrophysiology lab at California Polytechnic State University in San Luis Obispo is an example of a proven lab setup for high quality signal capture.

Pathogenic variants in KCNA2, encoding for the voltage-gated potassium channel Kv1.2, have been identified as the cause for an evolving spectrum of neurological disorders. Affected individuals show early-onset developmental and epileptic encephalopathy, intellectual disability, and movement disorders resulting from cerebellar dysfunction. In addition, individuals with a milder course of epilepsy, complicated hereditary spastic paraplegia, and episodic ataxia have been reported. By analyzing phenotypic, functional, and genetic data from published reports and novel cases, we refine and further delineate phenotypic as well as functional subgroups of KCNA2-associated disorders. Carriers of variants, leading to complex and mixed channel dysfunction that are associated with a gain- and loss-of-potassium conductance, more often show early developmental abnormalities and an earlier onset of epilepsy compared to individuals with variants resulting in loss- or gain-of- function. We describe seven additional individuals harboring three known and the novel KCNA2 variants p.(Pro407Ala) and p.(Tyr417Cys). The location of variants reported here highlights the importance of the proline(405)–valine(406)–proline(407) (PVP) motif in transmembrane domain S6 as a mutational hotspot. A novel case of self-limited infantile seizures suggests a continuous clinical spectrum of KCNA2-related disorders. Our study provides further insights into the clinical spectrum, genotype–phenotype correlation, variability, and predicted functional impact of KCNA2 variants.

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No description available.

In this study, four anion transporter (AT) blockers, DIDS (4, 4′-diisothiocyanatostilbene-2, 2′-disulfonic acid), 9-AC (anthracene-9-carboxylic acid), NPPB (5-nitro-2-(3-phenylpropylamino) benzoic acid), and IAA-94 (indanyloxy acetic acid) were selected to evaluate ATs as potential target sites for insect and nematode control. All the AT blockers showed slowly developing toxicity against second-stage larvae of <i>Meloidogyne incognita</i> (Kofoid and White 1919) Chitwood 1949 and adults of <i>Caenorhabditis elegans</i> Maupas 1900 but not against third-stage larvae of <i>Heterorhabditis bacteriophora</i> Poinar 1975 even at 200 ppm. Symptoms of AT blocker toxicity observed in <i>C. elegans</i> adults were increased pharyngeal muscle contractions and decreased locomotion. Exposure of <i>C. elegans</i> as fourth-stage larvae to double-stranded RNA (dsRNA) of <i>ceclc-1</i> and <i>ceclc-2</i> (VGCC genes coding for CeClC-1 and CeClC-2, respectively) either alone or together for 24 h decreased their expression in F1 progeny in a time-dependent manner. Reduction in expression of <i>ceclc-2</i> alone or together with <i>ceclc-1</i> significantly increased pharyngeal contractions and decreased locomotion in significantly higher percentage of F1 progeny. The above findings suggested AT blockers nematicidal activity primarily comes from inhibition of CeClC-2 channels, while inhibition of CeClC-1 channels may enhance this activity. All the AT blockers showed slowly developing toxicity against adults of a susceptible strain (Oregon-R) of <i>Drosophila melanogaster</i> Meigen 1830, while DIDS, was equally toxic to dieldrin-resistant rdl flies. All AT blockers, except 9-AC, at 100 µM showed significant excitatory effect on desheathed central nervous system (CNS) of third-instar larvae of <i>Drosophila</i>, while DIDS showed a modest excitatory effect on ascending peripheral nerves. Feeding adult flies on 10% sugar solution mixed with 100 ppm of DIDS for 6 h decreased the midgut pH by 2 units approximately. All the AT blockers inhibited the growth of larvae (in weight), increased the developmental time, and decreased survival when <i>Ostrinia nubilalis</i> (Hübner 1796) second-instar larvae were fed for seven days. All the AT blockers decreased the midgut alkalinity and inhibited chloride ion transport from midgut lumen into epithelia in fifth-instar larvae when fed for 3 h on treated diet. Positive correlations observed among growth, midgut alkalinity, and midgut chloride transport in AT blocker-fed larvae suggested that inhibition of chloride/bicarbonate exchangers by AT blockers may have contributed to midgut alkalinity decrease affecting the digestion and resulting in observed lethal and sublethal effects. / Ph. D.

Glass fiber based polymer composites based injection molded parts provide a light-weight high-strength alternative for use in automobile applications. These composites have enhanced mechanical properties compared to those of pure polymers, if the fibers are oriented in the right direction. One of the major challenges in processing of these composites is to control the fiber orientation in the final product. The evolution of short glass fiber orientation in a center-gated disk was experimentally determined along the radial direction at three different heights representative of the shell, transition and core layers, respectively. Orientation data along the shell and transition layers in the lubrication region show shear flow effects, which tends to align the fibers along the flow direction. In the core layer, where the extension in the "-direction dominates, fibers tend to get aligned along the "-direction. In the frontal flow region orientation in the flow direction drops in all three layers due to fountain flow effects. Fiber orientation predictions in coupled and decoupled transient simulations using the Folgar-Tucker model, and the two slow versions of the Folgar-Tucker model, namely the slip Folgar-Tucker model and the reduced strain closure (RSC) model were compared with the experimental data. Measured inlet orientation was used in all simulations and model parameters were determined by fitting model predictions to rheological data under startup of shear. Pseudo-concentration method was implemented for the modeling of the advancing front and fountain flow effects in the region near the front. Discontinuous Galerkin finite element method and a third order Runge-Kutta total variance diminishing time integration scheme were implemented for the solution of the orientation and transport equations. In the lubrication region of the shell layer, all three orientation models provided a good match with the experimental data. In the frontal region, fountain flow simulations showed characteristic features seen in r- and z-profiles of orientation, although the experimental data showed these features at a relatively larger distance behind the front while the simulations predicted these effects only up to a small distance behind the front. On the other hand, orientation predictions with the Hele-Shaw flow approximation showed significant over-predictions in the frontal region. With model parameters determined from fitting to rheological data, coupling did not show any significant improvements. However, with the use of a smaller value of the fiber interaction parameter, coupling showed significant improvement in orientation predictions in all three layers in the frontal region. The simulation scheme was extended to long fiber systems by comparing available long fiber orientation data in a center-gated disk with model predictions using the Bead-Rod model which considers fiber bending, a property exhibited by long semi-flexible fibers. The Bead-Rod model showed improvements over rigid fiber models in the lubrication region of the shell layer. However, close to the front, both models showed similar predictions. In fountain flow simulations, the flow features seen in the r- and z-profiles were much better predicted with both the models while Hele-Shaw flow approximation showed over-prediction of orientation in the flow direction, especially in the shell layer. / Ph. D.