Light entering the eye is transformed by the retina into electrical signals. Extensive processing takes place in the retina before these signals are transmitted to the brain. Beginning in the outer retina, light-evoked electrical signals are distributed into parallel pathways specialized for different visual tasks, such as the detection of dark vs. bright ambient light, the onset or offset of light, and the direction of stimulus motion. Pathway diversity is a consequence of cell type diversity, differential cell connectivity, synapse organization, receptor expression, or any combination thereof. Cell connectivity itself can be accomplished through excitatory or inhibitory chemical synapses, or electrical coupling via gap junctions. Gap junctions are further specialized based on the expression of different connexin subunit isoforms. In aggregate, this diversity gives rise to ganglion cells with highly specialized functions, including ON and/or OFF responses, contrast-tuning and direction-selectivity (DS). The directionally-selective circuit, a circuit specialized for the encoding of stimulus motion, makes use of many of these circuit specializations. Bipolar cells, in response to glutamate release from cone photoreceptors, provide highly-sensitive glutamatergic input to amacrine cells and DS ganglion cells (DSGCs) in this circuit, while amacrine cells provide cholinergic and directionally-tuned GABAergic input to DSGCs. One population of DSGCs also transmit signals laterally to one another via gap junctions. Thus numerous specializations in bipolar cells, amacrine cells and ganglion cells endow DSGCs with their unique encoding abilities. In Chapters 2 and 3 of this dissertation I focus on synchronized firing between gap junction-coupled DSGCs. sDSGCs exhibit fine-scale correlations, with action potentials in an sDSGC more likely within ~2ms of action potential firing in a coupled neighbour. I first characterize electrical coupling of DSGCs through the identification of the molecular composition of DSGC gap junctions (Chapter 2). Physiological and immunohistochemical methods allowed me to demonstrate an important role for connexin 36 subunits in DSGC electrical coupling. Next (Chapter 3) I investigate the sub-cellular mechanisms underlying neuronal correlations between electrically coupled DSGCs. Using paired recordings, I show that chemical input (from bipolar cells and amacrine cells), electrical input (from gap junctions), and Na+ channel activity in DSGC dendrites underlie the generation of correlated spiking activity. While a common feature of electrically coupled networks, the mechanisms underlying correlations were previously unclear. In Chapter 4 I focus on the mechanisms within the DS circuit that endow these neurons with impressive sensitivity to stimulus contrast. Using physiological and pharmacological methods I first assess the relative contrast sensitivity of ganglion cells and starburst amacrine cells (SACs) in the DS circuit. The sensitivity of DSGC and SAC excitatory currents to antagonists of Na+ channels suggests an important role for these channels in amplifying low contrast responses and other weak inputs to the circuit. This role is later attributed to the differential expression of voltage-gated Na+ channels in specific bipolar cell populations. In aggregate, this dissertation describes several novel circuit mechanisms within the well-studied DS circuit. I also provide specific roles for such specializations in visual coding. / Graduate

The amiloride-sensitive epithelial Na+ channel, ENaC, is the Na+-specific salt taste receptor in rodents. Compared to rodents, human salt taste perception is amiloride-insensitive. In rodents the ENaC is composed of aβg-subunits. Whereas humans express an additional subunit, the d-ENaC subunit. ENaC in human taste cells is composed of aβg-subunits or dβg-subunits, with the latter being amiloride-insensitive. Currently, it is not known if dβg-ENaC expression and trafficking is regulated by hormones and their downstream intracellular signaling effectors. The aim of this study is to investigate if arginine vasopressin (AVP), aldosterone, and cAMP regulate d-ENaC expression and trafficking in cultured fungiform human taste cells (HBO cells). Secondly, we want to demonstrate the expression of downstream signaling effectors involved in the trafficking of d-ENaC in HBO cells. Using molecular and immunocytochemical techniques, our results demonstrate that AVP, cAMP, and aldosterone increase expression of d-ENaC mRNA and protein in HBO cells. Furthermore, AVP, cAMP and aldosterone increased trafficking of the d-ENaC subunit from the cytosolic compartment to the apical pole of the HBO cells. Our results further demonstrate that HBO cells express several components of signaling cascade involved in ENaC translocation from cytosol to apical pole in HBO cells. The components of this signaling cascade include AVPR2, PKA, CREB, SGK-1, Nedd4-2, and GILZ-1. These hormones in mice and rats upregulate ENaC. Currently, we are not sure if these hormones affect ENaC this way in humans. By studying d-ENaC with these hormones, we are able to see how human ENaC is regulated in the tongue.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Increased electrical activity in peripheral sensory neurons contributes to pain. A unique type of sodium current, fast resurgent current, is proposed to increase nerve activity and has been associated with pain pathologies. While sodium channel isoform Nav1.6 has been identified as the main carrier of fast resurgent currents, our understanding of how resurgent currents are modulated in sensory neurons is fairly limited. Thus the goal of this dissertation was to identify resurgent current modulators. In particular, we focused on sodium channel beta subunits (Navβs) and fibroblast growth factor homologous factors (FHFs) in dorsal root ganglion (DRG) neurons. We hypothesized that Navβ4 and FHF2B act as positive regulators by mediating resurgent currents and modulating Nav1.6 inactivation, respectively. In contrast, we hypothesized FHF2A negatively regulates resurgent current by increasing the probability of channels in inactivated states. Thus, the aims of this dissertation were to 1) determine if Navβ4 regulates fast resurgent currents in DRG neurons, 2) examine the effects of Navβ4 knockdown on resurgent currents, firing frequency and pain associated behavior in an inflammatory pain model and 3) determine if FHF2A and FHF2B functionally regulate Nav1.6 currents, including resurgent currents in DRG neurons. To examine the aims, we used biochemical, electrophysiological and behavioral assays. Our results suggest that Navβ4 is a positive regulator of resurgent currents: in particular, the C-terminus likely mediates these currents. Localized knockdown of Navβ4 decreased inflammation-induced enhancement of resurgent currents and neuronal excitability, and prevented the development of persistent pain associated behavior in an inflammatory pain model. FHF2B increased resurgent currents and delayed inactivation. In contrast, FHF2A limited resurgent currents; an effect that is mainly contributed by FHF2A's N-terminus activity that increased accumulation of channels in inactivated states. Interestingly, in an inflammatory pain model FHF2B was upregulated and FHFA isoforms were downregulated. Together these results suggest that FHF2A/B modulation might contribute to enhanced resurgent currents and increased neuronal excitability observed in the inflammatory pain model. Overall, our work has identified three resurgent current modulators FHF2A, FHF2B and Navβ4. Manipulation of these proteins or their activity might result in novel strategies for the study and treatment of pain.

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

Many preterm infants require mechanical ventilation as life-saving therapy. However, ventilation-induced overpressure can result in lung diseases. Considering the lung as a viscoelastic material, positive pressure inside the lung results in increased hydrostatic pressure and tissue compression. To elucidate the effect of positive pressure on lung tissue mechanics and cell behavior, we mimic the effect of overpressure by employing an uniaxial load onto fetal and adult rat lungs with different deformation rates. Additionally, tissue expansion during tidal breathing due to a negative intrathoracic pressure was addressed by uniaxial tension. We found a hyperelastic deformation behavior of fetal tissues under compression and tension with a remarkable strain stiffening. In contrast, adult lungs exhibited a similar response only during compression. Young’s moduli were always larger during tension compared to compression, while only during compression a strong deformation-rate dependency was found. In fact, fetal lung tissue under compression showed clear viscoelastic features even for small strains. Thus, we propose that the fetal lung is much more vulnerable during inflation by mechanical ventilation compared to normal inspiration. Electrophysiological experiments with different hydrostatic pressure gradients acting on primary fetal distal lung epithelial cells revealed that the activity of the epithelial sodium channel (ENaC) and the sodium-potassium pump (Na,K-ATPase) dropped during pressures of 30 cmH2O. Thus, pressures used during mechanical ventilation might impair alveolar fluid clearance important for normal lung function.

No description available.

La restriction de croissance intrautérine (RCIU) est associée à l’apparition de maladies à l’âge adulte et le phénotype de la condition pathologique peut être différent selon le sexe. Notre laboratoire a développé un modèle de RCIU chez le rat en administrant une diète faible en sodium lors du dernier tiers de la gestation entraînant une réduction de l’expansion volémique maternelle et de la perfusion utéroplacentaire. L'activité rénine et la concentration d'aldostérone plasmatique sont augmentées chez la mère et les foetus RCIU. Antérieurement, notre laboratoire a démontré une augmentation de l’expression génique et protéique rénale de la Na+-K+-ATPase-α1 uniquement chez les foetus femelles RCIU. Ainsi, nous émettons l’hypothèse que la diminution du volume circulant chez la rate gestante entraîne une augmentation et une expression différentielle, selon le sexe, des éléments de la cascade de signalisation du récepteur des minéralocorticoïdes (MR) dans les reins de foetus RCIU. L’expression des gènes est réalisée par qRT-PCR et celle des protéines par immunobuvardage de type Western. Bien que les résultats démontrent que la transcription génique de SGK1, α-ENaC et GILZ soit augmentée dans les reins de foetus RCIU, l’expression protéique de SGK1, pSGK1(Thr 256) et α-ENaC est similaire à celle des témoins. La protéine GILZ est indétectable. Pour CNKSR3, aucune différence de l’ARNm ou de la protéine n’a été observée entre les deux groupes. Par contre, même si l’expression génique du MR n’est pas différente, l’expression protéique est diminuée chez les RCIU. Aucun effet du sexe n’a été observé. En conclusion, l’augmentation d'aldostérone plasmatique chez les foetus ayant subi une RCIU stimule la transcription des gènes associés à la voie de réabsorption sodique, mais la quantité protéique demeure inchangée. Ceci suggère qu’il peut avoir des mécanismes de régulation post-transcriptionnelle ou une dégradation accélérée des protéines. Malgré la pertinence du sexe dans le développement de maladies, le sexe n’influence pas l’expression des composantes de la voie de rétention sodique chez le foetus. Il serait important de suivre cette voie en fonction de l’âge et de corréler les expressions génique et protéique avec l’apparition de maladies. / Intrauterine growth restriction (IUGR) contributes to the development of diseases in adulthood, of which some are influenced by sex. Our laboratory has developed an IUGR model in the rat by administering a low-sodium diet during the last third of gestation which leads to a reduced volume expansion and uteroplacental perfusion. Plasma renin activity (PRA) and aldosterone concentration are increased in IUGR dams and foetuses. Previously, our laboratory has shown that gene and protein expression of Na+-K+-ATPase-α1 was increased solely in female IUGR kidneys. Therefore, we hypothesize that the reduced maternal volume will result in an increase and differential expression, influenced by foetal sex, of elements from the mineralocorticoid receptor (MR) pathway. Gene expression is determined by qRT-PCR and for protein, Western blotting is performed. Although results show that gene expression of SGK1, α-ENaC and GILZ is increased in IUGR foetal kidneys, protein expression for SGK1, pSGK1 (Thr256), and α-ENaC is similar to controls. GILZ protein is not detected. For CNKSR3, no difference in mRNA or protein expression is observed between the groups. Gene expression of MR is unchanged, while its protein expression is decreased in IUGR foetuses. In conclusion, the increase in plasma aldosterone in IUGR foetuses stimulates gene transcription of several components of the sodium reabsorption pathway without affecting protein expression. These results suggest that there may be post-transcriptional regulation or a higher protein turnover. Despite the importance of biological sex in the development of disease, it did not affect the expression of elements from the sodium reabsorption pathway in the foetus. It would be important to verify this same pathway in animals of different ages to correlate gene and protein expression with the appearance of diseases.

Helicoverpa armigera (Hübner) was officially reported in Brazil in 2013 causing serious damage to several crops, especially soybean and cotton crops. Because of this severe damage and also because H. armigera is more tolerant to insecticides in compare to other lepidopteran pests in Brazil, there was a significant increase of selection pressure with insecticides in the field. Many cases of insecticide resistance, especially to pyrethroids, have been reported in some countries of the Old World. The main objective of the present study was to characterize the susceptibility of H. armigera and to investigate the mechanisms of its resistance to pyrethroids and indoxacarb in Brazilian populations. Mortality of H. armigera populations was less than 50% when treated with the highest dose of 10 μg a.i./3rd-instar larva of fenvalerate and deltamethrin. Field populations of H. armigera monitored from 2013 to 2016 growing seasons showed mean mortalities of 10 to 40% at the diagnostic dose of 10 μg a.i./3rd-instar larva. The resistance ratio to pyrethroid was 780-fold. The frequency of the chimeric P450 CYP337B3 gene was above 0.95 in all 33 populations screened. The genetic basis of H. armigera resistance to pyrethroids was also investigated. The dominance degree varied from 0.66 to 0.92, i.e., incompletely to completely dominant, and resistance was characterized as autosomal and polygenic. Possible mutations in the sodium channel were investigated, as well as the expression of other P450 genes via RT-qPCR. Two non-synonymous mutations, V937G and Q960H were found, and the genes CYP6AB10, CYP301A, CYP4S13 and CYP321A5 were up-regulated in the Brazilian pyrethroid-resistant strain compared to the susceptible strain. The susceptibility of H. armigera populations to indoxacarb was characterized with a diet overlay bioassay in 3rd-instar larvae. LC50 values ranged from 0.22 (0.16-0.28) μg a.i./cm2 to 0.57 (0.41-0.82) μg a.i./cm2, varying 2.6-fold. The populations were monitored through the 2013-2017 growing seasons, with the diagnostic dose of 6.1 μg a.i./cm2; during the period, the susceptibility to indoxacarb decreased. An indoxacarb-resistant strain was selected under laboratory conditions and showed a resistance ratio of 297.5-fold. These results will contribute to decision-making and implementation of insect resistance-management (IRM) programs in Brazil and other recently invaded countries in Brazil. / Helicoverpa armigera (Hübner) foi reportada oficialmente no Brasil em 2013, ano em que causou grandes perdas em lavouras de soja e algodão no país. Devido ao ataque severo de H. armigera e por ser mais tolerante do que as demais pragas que ocorriam no Brasil, houve um aumento significativo da pressão de seleção com inseticidas no campo. Inúmeros casos de resistência desta praga a inseticidas do grupo dos piretroides já havia sido reportado em alguns países do Velho Mundo. Dentro desse contexto o objetivo desse trabalho foi caracterizar a suscetibilidade e investigar possíveis mecanismos de resistência a piretroides bem como indoxacarb no Brasil. A mortalidade das populações de H. armigera foi menor do que 50 % quando tratadas com a dose máxima de 10 μg i.a./lagarta de 3º instar para fenvalerato e deltametrina. As populações de campo de H. armigera monitoradas entre os anos de 2013 a 2016 na dose diagnóstica de 10 μg i.a./lagarta de 3º instar apresentaram mortalidade de 10 a 40%. A frequência do gene P450 CYP337B3 foi maior do que 0,95 em 33 populações testada. Além disso, as bases genéticas da resistência de H. armigera a piretroides foram investigadas e a razão de resistência com a linhagem suscetível foi de 780 vezes. O grau de dominância variou de 0,66 a 0,92, incompletamente e completamente dominante e a resistência foi caracterizada como autossômica e poligênica. Adicionalmente investigou-se a presença de possíveis mutações no canal de sódio bem como a expressão de outros genes P450 em uma linhagem resistente a piretroides. Foi possível detectar duas mutações não-sinonímias V937G, e Q960H no canal de sódio e os genes CYP6AB10, CYP301A, CYP4S13 e CYP321A5 foram super expressos na linhagem resistente. A suscetibilidade de populações de H. armigera para o inseticida indoxacarb foi caracterizada a partir de bioensaios de ingestão com lagartas de 3° instar. Os valores de CL50 variaram de 0,22 (0,16 - 0,28) μg i.a./cm2 até 0,57 (0,41 - 0,82) μg i.a./cm2 variando em 2,6 vezes. As populações foram monitoradas ao longo das safras agrícolas entre 2013 e 2017 com a concentração diagnóstica de 6,1 μg i.a./cm2 e observou-se uma diminuição na suscetibilidade da praga a indoxacarb. Uma linhagem resistente a indoxacarb foi selecionada em laboratório e comparada com uma linhagem suscetível de referência, apresentando uma razão de resistência de 297,5 vezes. Os resultados obtidos são extremamente importantes e poderão contribuir na tomada de decisões bem como na implementação de programas de manejo da resistência de insetos (MRI) no Brasil.

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.