The overall objective of this thesis was to examine mechanisms involved in the acute regulation of sarcoplasmic reticulum (SR) Ca2+-handling properties by second messenger signaling pathways in skeletal and cardiac muscle. The aim of the first study (Chapter Two) was to characterize changes in the kinetic properties of sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA) proteins in cardiac and skeletal muscles in response to b-adrenergic, Ca2+-dependent calmodulin kinase II (CaMKII) and protein kinase C (PKC) signaling. The aim of the second study (Chapter Three) was to determine if insulin signaling could acutely regulate SERCA kinetic properties in cardiac and skeletal muscle. The aim of the final study (Chapter Four) was to determine if alterations in plasma glucose, epinephrine and insulin concentrations during exercise are able to influence SR Ca2+-handling properties in contracting human skeletal muscle. Data collected in Chapter Two and Chapter Three were obtained using tissue prepared from a group of 28 male Sprague-Dawley rats (9 weeks of age; mass = 280 ± 4 g: X ± S.E). Crude muscle homogenates (11:1 dilution) were prepared from selected hind limb muscles (soleus, SOL; extensor digitorum longus, EDL; the red portion of gastrocnemius, RG; and the white portion of gastrocnemius, WG) and the left ventricle (LV). Enriched SR membrane fractions, prepared from WG and LV, were also analyzed. A spectrophotometric assay was used to measure kinetic properties of SERCA, namely, maximal SERCA activity (Vmax), and Ca2+-sensitivity was characterized by both the Ca50, which is defined as the free Ca2+-concentration needed to elicit 50% Vmax, and the Hill coefficient (nH), which is defined as the relationship between SERCA activity and Ca2+f for 10 to 90% Vmax. The observations made in Chapter Two indicated that b-adrenergic signaling, activated by epinephrine, increased (P<0.05) Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50), without altering Vmax in LV and SOL but had no effect (P<0.05) on EDL, RG, or WG. Further analysis using a combination of cAMP, the PKA activator forskolin, and/or the PKA inhibitor KT5270 indicated that the reduced Ca50 in LV was activated by cAMP- and PKA-signaling mechanisms. However, although the reduced Ca50 in SOL was cAMP-dependent, it was not influenced by a PKA-dependent mechanism. In contrast to the effects of b-adrenergic signaling, CaMKII activation increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 and increased nh, without altering SERCA Vmax in LV but was without effect in any of the skeletal muscles examined. The PKC activator PMA significantly reduced SERCA Ca2+-sensitivity, by inducing a right-shift in Ca50 and decreased nH in the LV and all skeletal muscles examined. PKC activation also reduced Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG and WG), but did not alter Vmax in LV or SOL. The results of Chapter Three indicated that insulin signaling increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50) and an increased nH, without altering SERCA Vmax in crude muscle homogenates prepared from LV, SOL, EDL, RG, and WG. An increase in SERCA Ca2+-sensitivity was also observed in enriched SERCA1a and SERCA2a vesicles when an activated form of the insulin receptor (A-INS-R) was included during biochemical analyses. Co-immunoprecipitation experiments were conducted and indicated that IRS-1 and IRS-2 proteins bind SERCA1a and SERCA2a in an insulin-dependent manner. However, the binding of IRS proteins with SERCA does not appear to alter the structural integrity of the SERCA Ca2+-binding site since no changes in NCD-4 fluorescence were observed in response to insulin or A-INS-R. Moreover, the increase in SERCA Ca2+-sensitivity due to insulin signaling was not associated with changes in the phosphorylation status of phospholamban (PLN) since Ser16 or Thr17 phosphorylation was not altered by insulin or A-INS-R in LV tissue. The data described in Chapter Four was collected from 15 untrained human participants (peak O2 consumption, VO2peak= 3.45 ± 0.17 L/min) who completed a standardized cycle test (~60% VO2peak) on two occasions during which they were provided either an artificially sweetened placebo (PLAC) or a 6% glucose (GLUC) beverage (~1.00 g CHO per kg body mass). Muscle biopsies were collected from the vastus lateralis at rest, after 30 min and 90 min of exercise and at fatigue in both conditions to allow assessment of metabolic and SR data. Glucose supplementation increased exercise ride time by ~19% (137 ± 7 min) compared to PLAC (115 ± 6 min). This performance increase was associated with elevated plasma glucose and insulin concentrations and reduced catecholamine concentrations during GLUC compared to PLAC. Prolonged exercise reduced (p<0.05) SR Ca2+-uptake, Vmax, Phase 1 and Phase 2 Ca2+-release rates during both PLAC and GLUC. However, no differences in SR Ca2+-handling properties were observed between conditions when direct comparisons were made at matched time points between PLAC and GLUC. In summary, the results of the first study (Chapter Two) indicate that b-adrenergic and CaMKII signaling increases SERCA Ca2+-sensitivity in the LV and SOL; while PKC signaling reduces SERCA Ca2+-sensitivity in all tissues. PKC activation also reduces Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG, and WG) but has no effect on Vmax in the LV and SOL. The results of the second study (Chapter Three) indicate that insulin signaling acutely increases the Ca2+-sensitivity of SERCA1a and SERCA2a in all tissues examined, without altering the Vmax. Based on our observations, it appears that the increase in SERCA Ca2+-sensitivity may be regulated, in part, through the interaction of IRS proteins with SERCA1a and SERCA2a. The results of the final study (Chapter Four) indicate that alterations in plasma glucose, epinephrine and insulin concentrations associated with glucose supplementation during exercise, do not alter the time course or magnitude of reductions in SERCA or Ca2+-release channel (CRC) function in working human skeletal muscle. Although glucose supplementation did increase exercise ride time to fatigue in this study, our data does not reveal an association with SR Ca2+-cycling measured in vitro. It is possible that the strength of exercise signal overrides the hormonal influences observed in resting muscles. Additionally, these data do not rule out the possibility that glucose supplementation may influence E-C coupling processes or SR Ca2+-cycling properties in vivo.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 5 uppsatser.

Adenoviruses have been extensively studied in the field of gene regulation, since their genes are subjected to a tightly controlled temporal expression during the virus lifetime. The early-to-late shift in adenoviral gene expression distinguishes two completely different programs in gene expression. The adenoviral L4-33K protein, which is the subject of this thesis, was previously implicated to be a key player in the transition from the early to the late phase of infection. Here we show that L4-33K activates late gene expression by functioning as a virus-encoded alternative RNA splicing factor activating splicing of transcripts containing weak 3’ splice sites; a feature common to the viral genes expressed at late times of infection. The splicing enhancer activity of L4-33K was mapped to a tiny arginine/serine (RS) repeat in the carboxyl-terminal domain of the protein. Also, the subcellular distribution to the nucleus with enrichment in the nuclear membrane and subnuclear redistribution to viral replication centers during a lytic infection was observed to depend on this motif. RS repeats are common features for the cellular splicing factors serine/arginine-rich (SR) proteins, which in turn are regulated by reversible phosphorylation. We further show that L4-33K is phosphorylated by two cellular protein kinases, the double-stranded DNA-dependent protein kinase (DNA-PK) and protein kinase A (PKA) in vitro. Interestingly, DNA-PK and PKA have opposite effects on the control of the temporally regulated L1 alternative RNA splicing. DNA-PK functions as an inhibitor of the late specific L1-IIIa pre-mRNA splicing whereas PKA functions as an activator of L1-IIIa pre-mRNA splicing. In summary, this thesis describes L4-33K as an SR protein related viral alternative splicing factor. A tiny RS repeat conveys splicing enhancer activity as well as redistribution of L4-33K to replication centers. Finally, DNA-PK and PKA that phosphorylates L4-33K are suggested to be novel regulatory factors controlling adenovirus alternative splicing.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

Orientadores: Everardo Magalhães Carneiro, Marcia Queiroz Latorraca / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T10:35:57Z (GMT). No. of bitstreams: 1 Veloso_RobertoVilela_D.pdf: 1133010 bytes, checksum: ae2bb8d027240ebc20854087fa364a1b (MD5) Previous issue date: 2007 / Resumo: A restrição protéica materna reduz o crescimento e promove alterações permanentes na estrutura e função de órgãos da prole, contribuindo para o desenvolvimento de doenças cardiovasculares, obesidade e diabetes. O consumo de alimentos à base de soja está associado a um menor risco de desenvolver diabetes pelo seu conteúdo de isoflavonas e pela composição de aminoácidos de sua proteína que contribuem para uma melhora na secreção de insulina. Tem sido sugerido que a genisteína, uma isoflavona da soja, modula a secreção de insulina através das vias AMPc/PKA e PLC/PKC. Deste modo, nós avaliamos o valor biológico da dieta à base de farinha de soja e seus efeitos sobre o crescimento de órgãos, o perfil de aminoácidos, insulina e metabólitos séricos em ratos submetidos à restrição protéica na infância e recuperados com essa dieta após o desmame, bem como o efeito da dieta à base de soja sobre a secreção de insulina em resposta a glicose e a ativadores do adenilato ciclase e proteína quinase C, além da expressão da PKAa e PKCa em ilhotas pancreáticas de ratos adultos. Ratos de mães alimentadas com 17% ou 6% de proteína (caseína) durante a gestação e lactação foram mantidos com dieta contendo 17% de proteína à base de caseína (grupos CC e RC) ou dieta com 17% de proteína à base de farinha de soja (grupos CS e RS) e dieta com 6% de proteína (grupo HP). Após 90 dias de idade, proles de mães alimentadas com dieta hipoprotéica exibiram déficit permanente de peso corpóreo e de concentrações séricas de insulina, taurina, glutamina, fenilserina e lisina, porém apresentaram um aumento no peso relativo dos órgãos, exceto do fígado. A dieta à base de farinha de soja reduziu o peso relativo do fígado e aumentou as concentrações séricas de insulina, taurina, ornitina e fenilserina. Embora ratos recuperados com soja (RS) tenham ingerido mais dieta proporcionalmente ao peso corpóreo do que os ratos recuperados com caseína (RC) eles mostraram menor coeficiente de eficácia alimentar, e resultou em peso corpóreo final similar entre esses grupos. As concentrações séricas de albumina e proteínas totais não diferiram entre os grupos RS e RC. A dieta à base de soja melhorou a resposta de células beta de ratos controles em concentrações fisiológicas de glicose, enquanto em ilhotas de ratos recuperados isso ocorreu na presença de concentrações suprafisiológicas de glicose. A presença de PMA induziu uma resposta secretória com potência similar em ilhotas dos grupos RS e CS e a expressão de PKC foi similar em todos os grupos, exceto no grupo HP, que expressou menores concentrações dessa proteína. A adição de forskolin ao meio de incubação aumentou a secreção de insulina em ratos recuperados e naqueles mantidos com caseína e a expressão de PKAa foi maior no grupo RS em relação ao grupo CS. Esses resultados sugerem que dieta à base de farinha de soja é capaz de promover a recuperação nutricional em animais submetidos à restrição protéica em fases críticas de desenvolvimento, melhorando o perfil de aminoácidos séricos que estimulam a secreção de insulina. Além disso, a melhora na secreção de insulina parece não ser devido a ativação das vias AMPc/PKA e inositol fosfato/PKC / Abstract: Maternal protein restriction leads to reduction in the growth of organs, permanent changes in their structure and functions contributing to development of cardiovascular disease, obesity and diabetes. Consumption of soy-based foods is associated to lower risk of diabetes by its isoflavone content and amino acid composition of its protein that contribute to improve of insulin secretion. It has been suggested that genistein, soy isoflavone, modulates the insulin secretion through of cAMP/PKA and PLC/PKC pathways. Thus, we evaluated the biological value of soybean flour diet and its effects on organ growth, serum amino acids, insulin and metabolites profile in rats submitted to protein restriction in early life and recovered with those diet after weaning, as well as, the effect of soybean diet on insulin secretion in response to glucose and activators of adenylate cyclase and PKC, besides the expression of PKA and PKC in pancreatic islets from adult rats. Rats from mothers fed with 17% or 6% protein (casein) during pregnancy and lactation were maintained with 17% casein (CC and CR groups) or soybean (SC and SR groups) diet and with 6% casein (LP groups) diet. At 90d of age offspring of protein-restricted-mothers exhibited permanent deficit of body weight, serum insulin, taurine, glutamine, phenylserine and lysine concentrations, but increased relative organs¿ weight, except liver. Soybean flour diet reduced the relative liver weight and increased serum insulin, taurine, ornithine and phenylserine concentrations. Although SR rats had eaten proportionally to body weight more diet than CR rats they showed lower feed conversion efficiency which resulted in the final body weight similar between these groups. The SR and CR also exhibited similar serum albumin and protein concentrations. Soybean diet improved the response of ß-cells from control rats to a physiological concentration of glucose, whereas in islets from recovered rats this occurred in presence of a supra-physiological glucose concentration. PMA induced similar potent secretory response in islets from SR and SC groups and PKC expression was similar in all groups, except LP that expressed lower levels this protein. Forskolin increased the insulin secretion in recovered rats and in those maintained with casein diet and PKA expression was higher in SR than in SC rats. These results suggest that soybean flour diet is able to promote the nutritional recovery in animals submitted to protein restriction in critical phase of development improving serum amino acid levels that have the stimulatory effect on insulin release. Moreover the improve of insulin secretion seemed does not to be due the activation of the cAMP/PKA and inositol phosphate/PKC pathways / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

L’objectif de cette thèse était de caractériser les éléments appartenant à la voie de signalisation énergétique AMP-activated protein kinase chez l’huître creuse Crassostrea gigas afin de comprendre son implication dans la gestion de l’énergie, en particulier en réponse à des conditions physiologiques qui sollicitent de l’énergie telles que la reproduction, ou à des stress environnementaux comme l’hypoxie ou le jeûne. Au niveau génomique, les trois sous-unités constitutives du trimère AMPK ainsi que plusieurs éléments impliqués dans cette voie de signalisation et dans les métabolismes glucidiques et lipidiques, potentiellement cibles de l’AMPK, ont été décrits. Au niveau protéique, plusieurs anticorps hétérologues ciblant les isoformes de la sous-unité α et la phosphorylation du résidu thréonine 172 de la sous-unité α, témoin indirect de l’activité AMPK, ont été utilisés. Deux sous-unités α tronquées dans le domaine kinase ont été caractérisées principalement dans les tissus musculaires suggérant leurs implications dans la fonction musculaire. Au cours d’un stress hypoxique, une augmentation significative des quantités de sous-unités α tronquées a été observée dans le muscle lisse. Ce résultat suggère que pendant une durée d’au moins 6 h, ces protéines tronquées sont nécessaires au maintien du métabolisme aérobie dans le muscle lisse, lui permettant ainsi de remplir son rôle de fermeture statique des valves. Nous avons suggéré une hypothèse indiquant que l’accumulation in vivo de ces sous-unitésα tronquées pourrait exercer un rôle de modulation ou de transdomination négative de l’activité de la sous-unité α entière. Dans la gonade, nous avons observé une activation de l’AMPK tout au long du processus de gamétogénèse afin de supporter les processus cataboliques de création de gamètes. Une diminution de cette activation a été observée lors du stade anabolique de mise en réserve des ovocytes. Enfin, lors d’un conditionnement en milieu contrôlé, une approche physiologique par privation de nourriture et une approche pharmacologique par injection d’AICAR ont été réalisées pour provoquer une modulation de l’AMPK. Les analyses ont montré que ni le jeûne ni l’AICAR n’ont induit une augmentation de la phosphorylation de la sous-unité α. Cependant, plusieurs changements liés à l’injection de l’AICAR ont été observés sur la physiologie de l’huître : la modification du rapport AMP:ATP chez les huîtres nourries en comparaison aux huîtres à jeun, et une mortalité dépendante de la dose injectée d’AICAR chez les huîtres mises à jeun. La caractérisation de l’AMPK chez C. gigas ouvre de nombreuses perspectives exigeant des études fonctionnelles poussées afin de démontrer le rôle pivot de cette kinase dans la gestion de l’énergie, comme démontré chez de nombreuses espèces de vertébrés, et ainsi décrypter le métabolisme énergétique de l’huître. / The objective of this thesis was to characterize elements implicated in the energypathway of the AMP-activated protein kinase of the Pacific oyster Crassostrea gigas. Thecharacterization of the elements was performed in the scope of understanding their involvementin energy management, particularly in response to physiological conditions requiring energy, asreproduction or environmental stress, such as hypoxia or fasting.At genomic level, the three subunits of AMPK trimer and several elements involved inAMPK signaling pathway and in carbohydrate and lipid metabolism, supposedly under AMPKcontrol, were described. Additionally, at proteomic level, several heterologous antibodiestargeting AMPKα subunit isoforms and threonine 172 phosphorylation site of AMPKα subunit,indirect witness of AMPK activity, were assayed. Two truncated α subunits in the kinase domainwere characterized essentially in muscles, suggesting their involvement in muscle function.During a hypoxic stress, a significant increase of truncated α subunits protein amount wasobserved in smooth muscle. These results suggest that, for a period of at least 6 h, thesetruncated subunits are necessary for the maintenance of aerobic metabolism in smooth muscle ofC. gigas, allowing it to fulfill its static closing valves. We suggested that in vivo accumulation oftruncated AMPKα could serve as modulator or as transdominant negative regulator of the fulllengthAMPKα activity. In the gonad, AMPK appeared to be activated through the process ofgametogenesis, in order to support the catabolic processes of gametes creation. During theanabolic phase, when oocyte reserves were created, a signal disruption was observed. Finally,during controlled experiment, a physiological approach by food deprivation and apharmacological approach using AICAR injections were performed to modulate AMPK signal.This analysis showed that neither fasting nor AICAR induced an increase of AMPKphosphorylation, as expected. Although, several changes related to AICAR injection wereobserved in oysters physiology, such as the change of the AMP:ATP ratio in fed oysters and aAICAR dose-related mortality in fasting oysters. AMPK characterization in C. gigas opens newperspectives demanding extensive functional studies to establish the key role of AMPK in energymanagement, as demonstrated in vertebrates’ species, in order to understand the oyster’s energymetabolism.

Rapidly growing tumor cells outgrow their blood supply resulting in a microenvironment with reduced oxygen and nutrients. Using an in vitro transformation model we found that cancer cells expressing the SV40 ST antigen (+ST cells) are more resistant to glucose deprivation-induced cell death than cells lacking the SV40 ST antigen (−ST cells). Mechanistically, we found that the ST antigen mediates this effect by activating a nutrient-sensing kinase, AMP-activated protein kinase (AMPK). We further show that AMPK mediates its effects, at least in part, by inhibiting mTOR (mammalian target of rapamycin), thereby shutting down protein translation, and by inducing autophagy as an alternate energy source. Resistance to anoikis upon anchorage-deprivation is yet another form of stress tolerated by both normal stem/progenitor cells of various tissues in our body and by cancer cells. Using mammospheres as a model to enrich for stem/progenitor cells we found that mammosphere formation is accompanied with increased activation of AMPK. Concomitant with AMPK activation, we detected increased phosphorylation of the anti-apoptotic protein PED/PEA15. We further demonstrate that AMPK directly interacts with and phosphorylates PEA15 at Ser116, thus establishing PEA15 as a new AMPK target. Thus, our study has identified AMPK-PEA15 signaling as a key component of sphere formation by both normal and cancerous breast tissues. During metastasis, epithelial cells lose attachments to their neighbors, acquire a mesenchymal-like morphology, a process termed as epithelial-mesenchymal transition (EMT) and become motile. Our results indicate that AMPK regulates EMT by both transcriptional and post-translational modification of EMT-inducing transcription factor, Twist. Thus, our study has identified a role for AMPK in nutrient deprivation, anchorage-independent growth, and epithelial-mesenchymal transition involved in metastasis. In addition, we have identified two novel substrates of AMPK, PEA15 and Twist, that may play key roles in cancer progression. Thus, our study suggests that targeting AMPK, or its newly identified substrates, can be explored as possible anti-cancer mechanisms.

1. The effect of urotensin II (U-II) on spontaneous transmitter release was examined in the frog to see if the biological activity of this vasoactive peptide extended to neural tissues. 2. In normal Ringer solution, frog and human U-II (fU-II and hU-II, respectively) caused concentration-dependent, reversible increases in miniature endplate potential (MEPP) frequency, with hU-II about 22 times more potent than fU-II. hU-II caused a dose-dependent increase in MEPP amplitude, whereas fU-II caused an increase, followed by a decrease with higher concentrations. 3. Increasing extracellular Ca 2+ three-fold had no effect on the MEPP frequency increase to 25 μM hU-II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca 2+ caused a 61% reduction in the MEPP frequency increase to 25 μM hU-II. 4. Pretreatment with the phospholipase C inhibitor U-73122 caused a 93% reduction in the MEPP frequency increase to 25 μM hU-II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5-trisphosphate (IP 3) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. 5. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 μM hU-II, but pretreating with protein kinase A inhibitors (H-89 and KT5720) reduced the MEPP frequency increase by 88% and completely abolished the increase in MEPP amplitude. 6. Our results show that hU-II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP 3 and cyclic AMP/protein kinase A.

No description available.

Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / Exposure to oxidative stress leads to accumulation of reactive oxygen species and this stimulates protective cellular functions as a compensatory response to prevent the spread of apoptotic signal and prevent cell death. The purpose of this dissertation is to understand the importance of PKCγ activation and regulation of the retinal gap junction protein Cx50, and what role PKCγ plays in this neuro-protective effect. Through electron microscopy we were able to show that PKCγ knockout mice retinas had incomplete cellular organization in the outer plexiform layer (OPL) of the retina, the layer of retina where Cx50 plays an important role in retinal cellular synapses. Electroretinograms confirmed that this structural disorganization also led to loss of functional response to light stimuli in PKCγ knockout mice retinas. In vivo exposure to 100% hyperbaric oxygen (HBO) caused significant degradation of the retina in knockout mice compared to control mice. Thicknesses of the inner and nuclear and ganglion cell layers were increased, with complete disruption of OPL in PKCγ KO mice retinas. Damage to the outer segments of the photoreceptor layer and ganglion cell layer was significantly more apparent in the central retinas of HBO-treated knockout mice. Cx50 immunolabeling showed significant reduction to HBO treatment of PKCγ control mice retinas, HBO treatment failed to produce reduction of Cx50 immunolabeling in KO mice retinas. In the R28 retinal cell line, PKCγ enzyme was shown to be activated by phorbol ester (TPA) and hydrogen peroxide. This resulted in translocation to the cellular membrane as confirmed by western blot and confocal microscopy. Suppression of PKCγ by siRNA rendered R28 cells more sensitive to oxidative stress-induced cell apoptosis, the process of apoptosis started earlier, and this resulted in cell death. R28 treatment with phorbol esters and hydrogen peroxide led to reduction in gap junction activity and Cx50 gap junction cell disassembly. This dissertation shows that PKCγ plays an important role in structural organization of retina and has a neuro-protective effect in response to oxidative stress, in part because of its control of Cx50.