Orientador: André Sampaio Pupo / Banca: Wilma de Grava Kempinas / Banca: Edson Antunes / Resumo: A ejaculação precoce é a disfunção sexual masculina com maiores taxas de incidência e prevalência. De etiologia complexa, as principais abordagens farmacológicas desta condição têm sido a utilização de anestésicos locais, inibidores seletivos da recaptura de serotonina e mais recentemente, inibidores da fosfodiesterase tipo 5. O reflexo ejaculatório é um processo altamente organizado com a interação de áreas centrais encefálicas e espinhais, centros autonômicos simpáticos e parasimpáticos além de reflexos somáticos. O sistema nervoso autônomo simpático, através da ativação de adrenoceptores 1 ( 1-ARs), particularmente o subtipo 1A, desempenha papel fundamental na fase de emissão do reflexo ejaculatório através da modulação da contratilidade de órgãos como o ducto deferente, vesícula seminal e próstata. Além disso, a participação do influxo de cálcio extracelular via canais de cálcio dependentes de voltagem tipo L (canais de cálcio tipo L) na atividade contrátil destes órgãos é bem caracterizada. Devido à importância dos 1-ARs e canais tipo L no reflexo ejaculatório, este trabalho avaliou a eficácia do antagonista de 1-ARs Tamsulosin e das diidropiridinas bloqueadoras de canais de cálcio Nifedipina e (S)-(+)-Niguldipina no retardo da ejaculação em ratos. Os resultados obtidos demonstram que a atividade contrátil do ducto deferente e vesícula seminal ao estímulo adrenérgico via ativação de 1-ARs in vitro é fortemente inibida pelo antagonista de 1-ARs Tamsulosin e pelas bloqueadoras de canais de cálcio Nifedipina e (S)-(+)-Niguldipina. Entretanto, embora a contração destes órgãos e, portanto, a fase de emissão do reflexo ejaculatório tenha sido afetada, a latência ejaculatória dos animais não foi alterada. Estes resultados indicam que o bloqueio de canais de cálcio tipo L ou o antagonismo de 1-ARs no ducto deferente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Premature ejaculation is one of the most common male sexual dysfunctions. With complex aetiology, the main pharmacological approaches involve local anesthetics, selective serotonin reuptake inhibitors and, more recently, phosphodiesterase 5 inhibitors. The sympathetic nervous system plays a crucial role in the ejaculation, controlling vasa deferentia, seminal vesicles and prostate contraction through 1- adrenoceptor ( 1-AR) activation. In addition, the contractions of these organs are dependent on influx of extracellular calcium through L-type voltage-dependent calcium channels (L-type calcium channels). Due to the involvement of 1-ARs and L-type calcium channels in the ejaculatory reflex, this dissertation evaluated the efficacy of the 1-AR antagonist Tamsulosin and of dihydropyridine calcium channel blockers Nifedipine and (S)-(+)-Niguldipine in the delay of ejaculation in rats. The results showed that the vas deferens and seminal vesicle contraction in response to 1-AR activation is potently inhibited by Tamsulosin, Nifedipine and (S)-(+)- Niguldipine. However, although vas deferens and seminal vesicles contractions have been inhibited, the ejaculatory latency in vivo was not affected. These results show that although L-type calcium channel blockade and 1-ARs antagonism in the vas deferens and seminal vesicle are effective in the inhibition of events related to the seminal emission phase of ejaculation, they are not able to delay the ejaculation. The present results indicate that modulation of contractions of organs involved in seminal emission is not a suitable strategy to retard of ejaculation / Mestre

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A Alpinia zerumbet à uma planta da famÃlia Zingiberaceae, denominada popularmente âcolÃniaâ no Nordeste do Brasil. à utilizada com fins medicinais no tratamento de hipertensÃo e tem sido estudada em relaÃÃo as suas propriedades farmacolÃgicas. Esta pesquisa teve por objetivo analisar o efeito farmacolÃgico das fraÃÃes hexÃnica (FHOEAz), clorofÃrmica (FCOEAz) e metanÃlica (FMOEAz) do Ãleo essencial da Alpinia zerumbet (OEAz) na reatividade vascular in vitro e nos parÃmetros cardiovasculares in vivo. O projeto de pesquisa foi aprovado pela ComissÃo de Ãtica em Pesquisa Animal, da Universidade Federal do CearÃ, sob os protocolos n 55/10 e n 18/2011, de acordo com as normas de boas prÃticas que envolvem o uso de animais experimentais. Para todos os experimentos foram utilizados ratos Wistar machos. Realizaram-se experimentos de reatividade vascular no banho de ÃrgÃos, com preparaÃÃes de aorta isolada de rato, com endotÃlio Ãntegro e desnudo, mantidos em carbogÃnio e soluÃÃo de Krebs-Henseleit com concentraÃÃo em mmol/L: NaCl: 118,0; KCl: 4,7; KH2PO4: 1,2; MgSO4.7H2O; 1,2; NaHCO3: 15,0; CaCl2: 2,5 e Glicose: 5,5. Foi observada a variaÃÃo da tensÃo isomÃtrica e utilizados diferentes fÃrmacos inibidores especÃficos para anÃlise do mecanismo de aÃÃo do efeito vasodilatador. Analisou-se a pressÃo arterial indireta por pletismografia de cauda em ratos submetidos ao modelo de hipertensÃo por inibiÃÃo crÃnica do Ãxido nÃtrico, atravÃs da administraÃÃo do L-NAME, obtendo-se a pressÃo arterial sistÃlica, diastÃlica e mÃdia, frequÃncia cardÃaca, alÃm da variaÃÃo do peso corporal. Constatou-se que o OEAz, FHOEAz, FCOEAz e FMOEAz induzem relaxamento de anÃis aÃrticos prÃ-contraÃdos com Fenilefrina (1 mol/L), de forma dependente da dose, sendo a menor CE50 a da FMOEAz (150,45 g/mL). A administraÃÃo por gavagem de 100 mg/kg de OEAz, FHOEAz, FCOEAz e FMOEAz reduziu a pressÃo arterial em ratos hipertensos pelo modelo de inibiÃÃo crÃnica do Ãxido nÃtrico, um efeito que foi superior ao controle negativo com Ãgua destilada e inferior aos controles positivos com Captopril e Nifedipina. A FMOEAz (0,1 - 3000 g/mL) induz relaxamento dependente da dose em anÃis aÃrticos prÃ-contraÃdos com Fenilefrina (1 mol/L) ou KCl (80 mmol/L), com endotÃlio intacto ou desnudo. Os estudos de mobilizaÃÃo de cÃlcio mostraram que a FMOEAz inibe o influxo de Ca2+ do meio extracelular, bem como interfere na contraÃÃo induzida pela liberaÃÃo de Ca2+ dos estoques intracelulares pela Fenilefrina (1 mol/L) ou CafeÃna (30 mmol/L). A 4-aminopiridina (1 mmol/L) e a Iberiotoxina (30 nmol/L) aumentam a CE50 da FMOEAz, mas nÃo interferem no seu efeito vasodilatador final. A prÃ-incubaÃÃo com L-NAME (100 mol/L), ODQ (10 mol/L); Indometacina (10 mol/L), Atropina (1 mol/L), Catalase (500 U/ml), SOD (300 U/mL); Wortmannina (0,5 mol/L), TetraetilamÃnio (10 mmol/L), Apamina (1 mol/L); Caribdotoxina (15 nmol/L) e Glibenclamida (10 mol/L) nÃo interferiram no relaxamento induzido pela FMOEAz. Concluiu-se que o OEAz, FHOEAz, FCOEAz e FMOEAz possuem efeito hipotensor in vivo e vasodilatador in vitro, e que o mecanismo de aÃÃo da FMOEAz, provavelmente, envolve o antagonismo aos canais de cÃlcio dependentes de voltagem, aos canais de cÃlcio operados por receptor, interferindo tambÃm na liberaÃÃo de cÃlcio dos estoques intracelulares. / The Alpinia zerumbet is a plant of the family Zingiberaceae, popularly called "colÃnia" in Northeastern Brazil. It is used for medicinal purposes to treat hypertension and has been studied in relation to its pharmacological properties. This study aimed to analyze the pharmacological effect of hexanic (HFEOAz), chloroformic (CFEOAz), and methanolic (MFEOAz) fractions of the essential oil of Alpinia zerumbet (EOAz) in the vascular reactivity in vitro and cardiovascular parameters in vivo. The research project was approved by the Ethics Committee on Animal Research, of the Federal University of CearÃ, under protocol numbers 55/10 and 18/2011, according to the standards of good laboratory practice involving the use of experimental animals. For all experiments male Wistar rats were used. Experiments of vascular reactivity were conducted in organ bath, with preparations of isolated rat aorta with intact endothelium and desnuded, kept in carbogen and Krebs-Henseleit solution with a concentration in mmol/L: NaCl: 118,0; KCl: 4,7; KH2PO4: 1,2; MgSO4.7H2O; 1,2; NaHCO3: 15,0; CaCl2: 2,5 e Glicose: 5,5. Was observed variation in isometric tension and used different specific inhibitors to analyze the mechanism of action of the vasodilator effect. We analyzed blood pressure indirectly by tail plethysmography in rats submitted to chronic hypertension by inhibition of nitric oxide by the administration of L-NAME, obtaining the systolic, diastolic and mean blood pressure, heart rate, beyond variation in body weight. It was found that the EOAz, HFEOAz, CFEOAz MFEOAz induced relaxation of aortic rings pre-contracted with Phenylephrine (1 mol/L), of dose-dependent manner, with the smallest of the EC50 MFEOAz (150.45 mg/mL). The administration by gavage of 100 mg/kg EOAz, HFEOAz, CFEOAz MFEOAz reduced blood pressure in hypertensive rats by the model of chronic inhibition of nitric oxide, an effect that was greater than the negative control with distilled water and less than the positive controls with Captopril and Nifedipine. The MFEOAz (0.1 - 3000 Âg/mL) concentration dependently relaxed Phenylephrine (1 mol/L) and KCl (80 mmol/L) contracted rings with intact or denuded endothelium. Studies of calcium mobilization showed that FMOEAz inhibits the influx of Ca2+ from the extracellular environment and interferes with the contraction-induced Ca2+ release from intracellular stocks by Phenylephrine (1 mol/L) or Caffeine (30 mmol/L). The 4-aminopyridine (1 mmol/L) and Iberiotoxina (30 nmol/L) increased the EC50 of MFEOAz but do not interfere in its final vasodilator effect. The pre-incubation with L-NAME (100 mol/L), ODQ (10 mol/L), Indomethacin (10 mol/L), Atropine (1 mol/L), Catalase (500 U/mL), SOD (300 U/mL), Wortmannin (0.5 mol/L), Tetraethylammonium (10 mmol/L), Apamin (1 mol/L); Caribdotoxin (15 nmol/L) and Glibenclamide (10 mol/L) did not interfere with the relaxation induced by MFEOAz. It was concluded that the EOAz, HFEOAz, CFEOAz and MFEOAz have hypotensive effect in vivo and vasodilator effect in vitro, and that the mechanism of action of MFEOAz probably involves the antagonism of calcium channels voltage-dependent, the calcium channel operated by receptor, also interfering in the release of calcium from intracellular stores.

Este estudo buscou compreender o canal de marketing usado para distribuição de servidores de microinformática do tipo x86 que, segundo institutos de pesquisas como o IDC (2009), responderão pela maioria absoluta de todos os servidores vendidos no país. Dentro da categoria de servidores x86, foram explorados os que estão em fases de crescimento e maturidade no ciclo de vida, devido aos potenciais de venda e de agregação de serviços ao cliente final. Para tanto, foram entrevistados doze membros deste canal, incluindo o fabricante dos produtos, três atacadistas, quatro varejistas e quatro clientes finais. Estes foram questionados sobre suas funções no canal de marketing analisado, avaliação de capacitação técnica para venda e prestação de serviços ao cliente, além dos papéis dos clientes e suas expectativas quando ao serviço ao cliente prestado. Percebeu-se que deficiência na capacitação e falta de estruturação nos varejistas pode ser um fator crítico para o canal de marketing, podendo comprometer a imagem dos produtos vendidos, o entendimento das necessidades e a prestação de serviços ao cliente e, de certa forma, o resultado de todos os outros membros do canal. / The intent of this study was to understand the marketing channel used to distribute x86 servers which, according to research institutes such as IDC (2009), will account for the majority of the servers sold in Brazil. Within the x86 category only products in growth or maturing lifecycle were considered in the investigation. These were selected due to potential of sales and customer services aggregation to the final purchasing customers. To accomplish that, twelve members of this channel were interviewed, including the product manufacturer, three wholesalers, four retailers and four final customers. They were asked about their function in the studied marketing channel, about their technical capability to sell and to serve customers, in addition to understanding the customer roles and their expectations on the performed service. The results found that reseller deficiency in technical skills and poor structure definition may be a critical factor for the marketing channel. This possibly compromises the image of sold products, the understanding of customer needs, customer services delivered and, in a certain way, the results for the other channel members.

Corticotroph cells from the anterior pituitary are an integral component of the hypothalamic-pituitary-adrenal (HPA) axis, which controls the neuroendocrine response to stress. Following stressful stimuli, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) from the hypothalamus act synergistically to stimulate adrenocortiotrophin hormone (ACTH) secretion from corticotroph cells. ACTH is released into the circulation where it stimulates the secretion of glucocorticoids from the adrenal cortex. The HPA axis is kept in fine balance through an elegant negative feedback system where elevation of plasma glucocorticoids results in inhibition at the level of both the pituitary and the hypothalamus. During acute stress, glucocorticoids can be beneficial however chronic elevation of glucocorticoids can have many adverse effects on health. Corticotroph cells are electrically excitable and have been shown to fire single-spike action potentials as well as complex bursting patterns. Stimulation of corticotrophs with physiological concentrations of CRH/AVP results in a robust increase in firing frequency and a transition from spiking to bursting. Intracellular Ca2+ increases to a greater extent during bursting which has been proposed to drive hormone secretion. There is evidence to suggest that large conductance calcium- and voltage-gated potassium (BK) channels promote bursting behaviour in anterior pituitary cells. Glucocorticoids have been shown to regulate ACTH secretion and also modulate BK channel activity. However, the effects of glucocorticoids on native corticotroph excitability are currently unknown. The aim of this study was to first characterise the electrical properties of corticotrophs under basal conditions and following exposure to CRH/AVP. Secondly, to investigate the regulation of corticotroph excitability by glucocorticoids. Finally, establish the role of the BK channel in regulating bursting behaviour and CORT regulation in corticotroph cells. Corticotroph cells were acutely isolated by trypsin digestion from mice aged 2-5 months constitutively expressing GFP under control of the POMC promoter (POMC-GFP). Mice used for pituitary cell culture were male unless otherwise stated. Cells were maintained in a serum free media and electrophysiological recordings obtained 24-96 hours post-isolation. Current clamp recordings were obtained from corticotrophs using the perforated patch technique. Although spontaneous activity of corticotroph cells was variable, they displayed predominantly single-spike action potentials under basal conditions. Stimulation with physiological concentrations of CRH and AVP (0.2 nM and 2 nM respectively) resulted in a membrane depolarisation accompanied by an increase in firing frequency and a transition to bursting. Individually, CRH and AVP were able to increase corticotroph excitability. However, only CRH was able to drive an increase in bursting suggesting that bursting is primarily regulated through the cAMP/PKA pathway. Experiments were performed to investigate the modulation of corticotroph activity by glucocorticoid negative feedback. Acute exposure (< 10 min) to corticosterone resulted in a decrease in spontaneous activity as well as shortening the response to CRH/AVP. Pretreatment of corticotrophs with 100 nM corticosterone (90 min) resulted in a membrane hyperpolarisation and a decrease in spontaneous firing frequency. Following corticosterone pretreatment, CRH/AVP failed to induce a significant transition from spiking to bursting. Increasing the pretreatment time to 150 minutes resulted in a further suppression of both spontaneous and CRH/AVPevoked activity. Fast activation of BK channels during the upstroke of an action potential has been proposed to promote bursting behaviour in other pituitary cells. Corticotrophs treated with a BK channel blocker (1 μM paxilline) or isolated from BK-/- mice showed no significant difference in basal activity but displayed a reduction in CRH/AVPevoked bursting activity. In both cases, bursting was significantly reduced but not completely abolished. Corticosterone treatment of BK-/- cells resulted in a further decrease in both firing frequency and bursting behaviour. Taken together, these results suggest that although BK channels play an important role in bursting, they are not the only component. Comparisons of male and female corticotrophs revealed subtle differences in their properties. Following CRH/AVP stimulation, male cells displayed a high degree of bursting activity whereas female cells exhibited predominantly an increase in singlespike action potential frequency. Treatment of female corticotrophs with corticosterone (150 min) resulted in a significant reduction in firing frequency but no measurable change in bursting behaviour. BK-/- cells from female mice showed no difference in bursting activity following CRH/AVP compared to wild types. This data suggests that modulation of firing frequency is the more important component in female corticotroph cells. In conclusion, CRH/AVP is proposed to drive ACTH secretion in male corticotroph cells through an increase in bursting activity. Corticosterone pretreatment suppresses both spontaneous and CRH/AVP-evoked activity. It is possible that corticosterone regulates corticotroph excitability through two mechanisms. Corticosterone suppresses bursting activity following CRH/AVP stimulation through multiple targets which might include the BK channel. Additionally, corticosterone reduces firing frequency through a mechanism independent of BK channels. It is important to further characterise the physiology of corticotroph cells and how ACTH secretion is regulated through their electrical excitability. This would lead to a greater understanding of the role of corticotrophs in the HPA axis. Further study of corticotrophs could potentially lead to pharmacological manipulation of the stress response and novel treatments for stress-related disorders.

Calmodulin (CaM) is an essential protein found in all eukaryotes ranging from vertebrates to unicellular organisms such as Paramecia. CaM is a calcium sensor protein composed of two domains (N and C) responsible for the regulation of numerous calcium-mediated signaling pathways. Four calcium ions bind to CaM, changing its conformation and determining how it recognizes and regulates its cellular targets. Since the discovery of CaM, most studies have focused on the role of its calcium-saturated form. However, an increasing number of target proteins have been discovered that preferentially bind apo (calcium-depleted) CaM. My study focused on understanding how apo CaM recognizes drugs and protein sequences, and how those interactions differ from those of calcium-saturated CaM. I have used spectroscopic methods to explore CaM binding the drug Trifluoperazine (TFP) and the IQ-motif of the type 2 Voltage-Dependent Sodium Channel (Nav1.2IQp). These studies have shown that both TFP and Nav1.2IQp preferentially bind to the "semi-open" conformation of apo CaM. TFP was shown to be an unusual allosteric effector of calcium binding to CaM. Using 15N-HSQC NMR spectroscopy, I determined the stoichiometry of TFP binding to apo Cam to be 2:1 and to (Ca2+)4-CaM to be 4:1 TFP:CaM. That difference in stoichiometry determined whether TFP decreased or increased the affinity of CaM for calcium. Analysis of residue-specific chemical shift differences indicated that TFP binding to apo and (Ca2+)4-CaM perturbed the C-domain more than the N-domain, prompting high-resolution structural studies of the isolated C-domain of CaM. Crystallographic studies of TFP bound to a calcium-saturated C-domain fragment of CaM (CaM76-148) revealed that CaM adopted an "open" tertiary conformation. The unit cell contained two protein and 4 drug molecules. The orientation of TFP revealed that its trifluoromethyl group was found in two alternative positions (one in each protein in the unit cell), and that Met 144 acted as a gatekeeper to select the orientation of TFP. In contrast to TFP binding to the "open" conformation of calcium-saturated CaM76-148, my NMR studies showed that TFP bound the "semi-open" conformation of apo CaM76-148. TFP interacted with CaM residues near the perimeter of the hydrophobic pocket, but did not contact residues that are solvent-accessible only in the "open" form. Allosteric effects due to TFP binding were observed in the calcium-binding loops of apo CaM76-148. These properties suggest that TFP may antagonize interactions between apo CaM and target proteins such as ion channels that preferentially bind apo CaM. Nav1.2, is responsible for the passage of Na+ ion across cellular membranes. Apo binding of CaM to Nav1.2 poises it for action upon calcium release in the cell. My NMR studies of CaM binding to the Nav1.2 IQ-motif sequence (Nav1.2IQp) showed that the C-domain of apo CaM was necessary and sufficient for binding. My high-resolution structure of the isolated C-domain of CaM bound to Nav1.2IQp revealed that the domain adopted a "semi-open" conformation. At the interface between the IQ-motif and CaM, the highly conserved I and two Y residues of Nav1.2IQp interacted with hydrophobic residues of CaM, while the invariant Q residue interacted with residues in the loop between helices F and G of CaM. This is the first CaM-IQ complex to be determined by NMR; the only other available structure of apo CaM bound to an IQ-motif was determined crystallographically. To accomplish its regulatory roles in response to cellular Ca2+ fluxes, CaM has evolved multiple binding interfaces that are allosterically linked to its Ca2+-ligation state. My studies of CaM binding to TFP and NaV1.2 demonstrate the versatility of CaM functioning as a regulatory protein comprised of domains having separable functions.

Proper control of movements is critical for an animal’s survival, and requires the robust function of a number of genetic, molecular, neuronal and biomechanical processes. This dissertation describes a body of inter-related studies utilizing a diverse collection of Drosophila mutants to probe the roles individual genes play in shaping motor pattern generation. A particular emphasis is placed on describing the consequences of genetic perturbations of voltage-gated sodium, calcium and potassium ion channels (NaV, CaV, and KV respectively) on the function of neuronal circuits that drive motor behavior. Here, I describe the development of several quantitative protocols to study alterations in of walking (IowaFLI Tracker) and flight motor program activity and behavior in Drosophila mutants. These approaches were utilized to analyze the highly-stereotypic aberrant motor program associated with electroconvulsive stimulation (ECS)-induced seizure discharge activity in each hyperexcitable mutant. Several quantitative and mechanistic similarities between flight motor program activity and ECS-evoked discharges were identified, and the distinct aberrant ECS-evoked activity disclosed an electrophysiological signature of each mutation. Ion channel mutants display a diverse spectrum of neuronal excitability phenotypes that was highlighted in a novel hyperexcitable mutant, Shaker wings down (Swd), characterized by ether-induced leg shaking reminiscent of certain KV channel mutants (e.g. Shaker, KV1) is presented. Detailed analyses revealed disrupted walking and flight, correlated with neuronal hyperexcitability and aberrant action potential generation. Surprisingly, the Swd mutation site was mapped to a single amino acid in the voltage sensor region in paralytic (para, encoding the only NaV gene in Drosophila). Genetic analysis of intra-genic heteroallelic interactions amongst Swd and other identified para alleles further revealed a number of complex mechanisms underlying a wide phenotypic spectrum of altered neuronal excitability and motor pattern generation. The effects of perturbed ion channel function on motor program generation are compared with progressive alterations associated normal aging as well as neurodegeneration. A number of age-resilient and age-vulnerable circuits were identified along with circuit-function biomarkers of aging. Throughout this study, an integrative framework utilizing non-linear dimensional reduction approaches unraveled a broader perspective to visualize and quantify similarities and distinctions between discharge phenotypes across a large collection of Drosophila mutants.

Cell surfaces are replete with complex, biologically important glycans responsible for multiple cellular functions including cell adhesion and cellular communication. Proper protein glycosylation is essential for normal development and often pathologies are marked by altered glycosylation. Here, data showed that the auxillary subunit, ß1, modified voltage-gated Na+ channel (Nav) gating in an isoform-specific, sialic acid dependent, and saturating manner. The regulated activity of the hundreds of glycogenes (glycosylation-associated genes) is responsible for protein glycosylation; this could result in a glycome of thousands of glycan structures. Microarray analyses indicated that glycogene expression was highly regulated throughout the heart during development. Specifically, >59% of glycogenes were significantly differentially expressed among neonatal and adult atrial and ventricular myocytes. Quantitative-PCR of individual genes confirmed the microarray analyses. Such substantial regulation of glycogene expression likely results in changes in glycan structures attached to cell surface proteins. To confirm this, myocyte glycan profiles were determined and compared among neonatal and adult atria and ventricles using mass spectrometry. The data predicted marked differences in glycan structures among myocyte types, indicating that the glycome is remodeled throughout the heart during development. To address the question of whether the remodeled glycome can impact cardiac function, action potentials and Na[subscript]v activity were measured and compared under conditions in which glycogene expression was regulated. That is, atrial and ventricular myocytes were isolated from control mice and from mice in which the polysialyltransferase, STX, was knocked out. STX is expressed in the neonatal atria, and is essentially absent in neonatal ventricle. Action potential waveforms and Nav activity measured in atrial myocytes were impacted by STX expression. No changes in ventricular action potential waveform or in Na[subscript]v activity were observed; as expected since STX is not expressed in the ventricle. The magnitude of the atrial action potential and the rate of depolarization were decreased in the absence of STX. Further, Na[subscript]v gating was shifted consistently in the depolarized direction in STX knockout atrial myocytes. Together, these data indicate that the glycome is tightly controlled and regulated in the heart, and proper glycosylation is essential for normal myocyte function.

The most important and most common channels on cell membrane are voltage-gated Na+ and K+ channels. In so-called "excitable cells" like neurons and muscle cells, these channels open or close in response to changes in potential across the membrane in order to accomplish muscle contraction and transmit signals. By controlling the membrane potential, we observe extraordinary inactivation behaviors of the voltage-gated Na+ channels and the voltage-gated delayed rectifier K+ channels, which shows that electric stimulation pulses can temporarily close the Na+ and K+ channels, just as drugs, like tetrodotoxin (TTX) and tetraethylammonium (ETA), do. The Na/K pump is essential for living system and is expressed in virtually all cell membranes. The ionic transport conducted by Na/K pumps creates both an electrical and a chemical gradient across the plasma membrane, which are required for maintaining membrane potentials, cell volume, and secondary active transport of other solutes, etc. We use a pulsed, symmetric, oscillating membrane potential with a frequency close to the mean physiological turnover rate across the cell membrane to synchronize Na/K pump molecules. The pump molecules can work as a group, pumping at a synchronized pace after a long train of pulses. As a result, the pump functions can be significantly increased. After the pump molecules are synchronized, the applied electric-field frequency can gradually increase in order to resynchronize the molecules to a new, higher frequency. Modulating the pump molecules to a higher frequency leads to a significant increase of pump current. Synchronization and modulation of pump molecules can become a new method to study the function of Na/K pump molecules. This method has huge potential applications in clinic medical treatment. After single-fiber-level study, the final project is on organ level, the rat kidney, by using synchronization and modulation of Na/K pump molecules on the proximal tubule membrane. Because Na+ re-absorption is directly related to the function of the Na/K pump, the more active Na/K pumps are, the more Na+ ions can be absorbed, which results in an increased potential inside the renal proximal tubule. This project is the first step of synchronization and modulation applied on the level of an organ.

Changes in the amount of water and sediment that enter a river can change its shape and size. The way that rivers change is affected by a variety of factors, including the size of the sediment in the river, and past changes to the river. The Diamond Fork River in central Utah has been altered by water deliveredfromthe Colorado River system for over a century. Beginning in 1915, water used for irrigation was delivered through a tributary, Sixth Water Creek, with daily summer flows that were much larger than natural flows. This caused drastic change to the rivers, as they became wider and vegetation along the channel margin and floodplain was destroyed. Management changes in 1997 and 2004 reduced the amount of water and sediment added to the river. In this study, we sought to understand how Sixth Water and Diamond Fork changed in the past and what the implications are for the future. We used data from a variety of sources to describe how and why the river changed in the past. Our results indicate that parts of the river that are not confined by valley walls became very wide during the period of elevated flows and narrowed after the change in management in 1997. Confined reaches experienced minor changes over the period of record. Areas of the channel that were most dynamic in the past are the most susceptible to future change because they have finer sediment that is more easily erodible. Areas that did not experience past changes are unlikely to change in the future without direct intervention from humans or beaver. The findings of this study improve our understanding of Sixth Water and Diamond Fork, and confirm the importance of past changes and valley confinement.

Modern processors use architecture like caches, branch predictors, and speculative execution in order to maximize computation throughput. For instance, recently accessed memory can be stored in a cache so that subsequent accesses take less time. Unfortunately microarchitecture-based side channel attacks can utilize this cache property to enable unauthorized memory accesses. The Spectre attack is a recent example of this attack. The Spectre attack is particularly dangerous because the vulnerabilities that it exploits are found in microprocessors used in billions of current systems. It involves the attacker inducing a victim’s process to speculatively execute code with a malicious input and store the recently accessed memory into the cache. This paper describes the previous microarchitecture side channel attacks. It then describes the three variants of the Spectre attack. It describes and evaluates proposed defenses against Spectre.