Les leucémies aigües lymphobastiques (LAL) représentent un tiers des leucémies et constituent le cancer pédiatrique le plus fréquent chez l’enfant. Les LAL de type T (LAL-T)sont caractérisées par l’expansion anormale de progéniteurs de lymphocytes T. Aujourd’hui,la réponse curative aux traitements est proche de 80% chez l’enfant et 50% chez l’adulte. La rechute reste donc fréquente et souvent de mauvais pronostic. Pour ces raisons,l’identification de nouvelles voies de signalisation en vue de développer de nouvelles stratégies thérapeutiques est cruciale afin d’améliorer le traitement des LAL-T.Les résultats précédents du laboratoire ont révélé l’activation soutenue de la voie calcineurine (Cn)/NFAT dans des échantillons humains de lymphomes et de LAL, ainsi que dans des modèles murins de ces pathologies. Le laboratoire a ensuite montré que Cn est intrinsèquement requise pour la capacité des cellules leucémiques de LAL-T à propager la maladie (activité LIC « Leukemia Initiating Cells ») dans un modèle murin de LAL-T induit parun allèle activé de NOTCH1 (ICN1). Puisque l’inhibition pharmacologique de Cn induit de nombreux effets secondaires, la recherche de cibles thérapeutiques en aval de Cn constitue un axe de recherche important. J’ai participé à une étude du laboratoire montrant que l’expression à la surface cellulaire de CXCR4 est régulée par Cn et requise pour la migration des cellules de LAL-T, mais non suffisante pour rétablir le potentiel de ré-initiation suggérant que d’autres effecteurs doivent être impliqués dans cette activité.Les facteurs de transcription NFAT (NFAT1, NFAT2 et NFAT4) sont des effecteurs importants de Cn en réponse à la signalisation calcique lors du développement des thymocytes, mais également dans les lymphocytes T. L’essentiel de ce travail de thèse a utilisé des LAL-T induites par ICN1 dans lesquelles l’inactivation génique des trois facteurs NFAT par recombinaison homologue. Nous avons ainsi montré que (i) les facteurs NFAT sont requis en aval de Cn pour le potentiel LIC des LAL-T-ICN1 in vivo, (ii) leur inactivation altère la survie, la prolifération et la migration des cellules de LAL-T in vitro, (iii) NFAT1,NFAT2 et NFAT4 ont une fonction largement redondante dans les LAL-T. Nous avons également par une approche transcriptomique identifié deux gènes dont l’expression estsous contrôle des facteurs NFAT et impliqués dans la régulation de la survie et de la prolifération des LAL-T in vitro : CDKN1A et MAFB.Tout comme la voie Cn/NFAT, les CaMKs sont des protéines kinases activées en aval de la signalisation calcique dans les lymphocytes T. Nous avons montré par une approche pharmacologique que l’inhibition des CaMKs dans les LAL-T-ICN1 in vitro altère la survie etla prolifération des cellules leucémiques. L’inhibition spécifique par une approche d’ARN interférence de deux isoenzymes CaMKIIγ et CaMKIIδ suggèrent que ces protéines jouent dans le maintien des cellules leucémiques in vitro. / T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of T cell progenitors. Despite initial response to chemotherapy, relapses remain frequent in children and adults. Previous results identify sustained activation of Calcineurin (Cn)/NFAT signaling pathway in human T-ALL and murine T-ALL models. Importantly, they also demonstrated Cn is essential for T-ALL Leukemia Initiating Cells (LIC) activity in a murine model of T-ALL induced by an activated allele of NOTCH1 (ICN1). Since pharmacologic inhibition of Cn induces side effects, we aim to identify downstream effectors involved in T-ALL. NFAT (Nuclear Factor of Activated T cells) factors play crucial roles downstream Cn during development and activation of T cells. To address their role in T-ALL, we generated mouse ICN1-induced T-ALL in which NFAT genes can be inactivated either single or in combination following Cre-mediated gene deletion. We demonstrated that (i) NFAT factors are required downstream Cn for LIC activity in T-ALL in vivo (ii) ex vivo NFAT factors deletion alters survival, proliferation and migration of T-ALL (iii) NFAT1, 2 and 4 have a largely redundant function in T-ALL. Moreover, the NFAT-dependant transcriptome allowed to identify important targets (CDKN1A, MAFB) involved in T-ALL survival and proliferation in vitro. Calmodulin-dependant kinases (CaMK) are kinases activated by calcium signaling in T cells. We showed that pharmacologic inhibition of CaMKs in ICN1-induced T-ALL alters survival and proliferation of T-ALL in vitro. Beside, specific inhibition by RNA interference of CaMKIIg and CaMKIId suggests a putative role of these kinases in T-ALL maintenance.

Orientador: Cristina Antoniali Silva / Banca: Fernando Silva Carneiro / Banca: Carlos Alan Candido Dias Junior / Banca: Graziela Scalianti Ceravolo / Banca: Angela de Castro Resende / Resumo: A redução da reatividade vascular à fenilefrina (PE) em aorta de ratas espontaneamente hipertensas (SHR) ao final da prenhez é dependente de maior produção e/ou maior biodisponibilidade de óxido nítrico (NO), consequente do aumento da fosforilação da enzima óxido nítrico sintase endotelial (eNOS) via PI3K/Akt. A glicosilação do tipo N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-traducional que compete com a fosforilação pelos mesmos sítios de ligação nas proteínas. A O-GlcNAcilação da eNOS em serina1177 leva a redução da sua atividade enquanto a fosforilação leva a sua ativação. Além destes mecanismos, a interação da eNOS com outras proteínas é capaz de regular positiva ou negativamente a sua atividade. O objetivo deste trabalho foi analisar possíveis alterações nos mecanismos de modificação pós-traducional que controlam a ativação da eNOS os quais poderiam contribuir para maior ativação e maior biodisponibilidade de NO observada em artérias de ratas prenhes. Foram avaliados o conteúdo proteico O-GlcNAc e também expressão das enzimas que participam desta modificação, O-GlcNAc transferase (OGT) e O-GlcNAcase (OGA) por Western Blotting e a atividade da OGA por ensaio bioquímico em aorta e em artéria mesentérica (2º ou 3º ramo) de ratas não prenhes (NP) e prenhes (P), normotensas (Wistar) e SHR. Ensaios de Western Blotting foram realizados também para análise da expressão das seguintes proteínas: Cav-1, p-Cav-1, CaM e Hsp90. Realizamos a contagem do número de cavéolas en... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Reduction of vascular reactivity to phenylephrine (PE) in aortaof spontaneously hypertensive rats (SHR) at the end of pregnancy is dependent on higherproduction and/or higerbioavailability of nitric oxide (NO), as a consequence of increased endothelial nitric oxide synthase enzyme (eNOS) phosphorylation,by PI3K/Akt.Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc)is a post-translational modification that competes with phosphorylation by the same binding sites in proteins. O-GlcNAcylation of eNOSon serine siteleads to a reduction in its activity while eNOS phosphorylation leads to its activation. In addition to these mechanisms, the interaction of eNOS with other proteins is able to regulate positively or negatively its activity. The objective of this studywas to analyze possible changes in the mechanisms of post-translational modification that control the eNOS activation, which could contribute to its the greater activation and greater bioavailability of NO observed in arteriesof pregnant rats. The O-GlcNAc-protein content and also the enzymesexpressionthat participate in this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) was assessed by Western Blotting, and OGA activity were evaluated by biochemical assay in the aorta and in the artery mesenteric (2ndor 3rdbranch) of non-pregnant (NP) and pregnant (P), normotensiverats(Wistar) and SHR.Western Blotting assays were also performed for expression analysis of the following proteins: Cav-1, p-Cav-1, CaM and Hsp90. We performed the counting of the number of endothelial caveolaein the aorta and the mesenteric artery in the presence or absence of methyl-β-cyclodextrin (dextrin, 10 mmol/L) by electronicmicroscopy.In functional studies, we evaluated the participation of the OGA enzyme, by inhibition with PugNAc (100 μmol/L) and of the caveolae, using a caveolae disassembler, (Complete abstract electronic access below) / Doutor

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.

The developmental processes of lymphopoiesis generate mature B lymphocytes from hematopoietic stem cells through increasingly restricted intermediates. Networks of transcription factors regulate these cell fate choices and are composed of both ubiquitously expressed and B lineage-specific factors. E-protein transcription factors are encoded by the three genes E2A, E2-2 (SEF2-1), and HEB. The E2A gene is required for B cell development and encodes the alternatively spliced proteins E12 and E47. During B lymphocyte development, the cells have to pass several checkpoints verifying the functionality of their antigen receptors. Early in the development, the expression of a pre-B cell receptor (pre-BCR) with membrane-bound immunoglobulin (Ig) heavy chain protein associated with surrogate light chain (SLC) proteins is a critical checkpoint that monitors for functional Ig heavy chain rearrangement. Signaling from the pre-BCR induces survival and a limited clonal expansion. Here it is shown that pre-BCR signaling rapidly down-regulates the SLCs l5 and VpreB and also the co-receptor CD19. Ca2+ signaling and E2A were shown to be essential for this regulation. E2A mutated in its binding site for the Ca2+ sensor protein calmodulin (CaM), and thus with CaM-resistant DNA binding, makes l5, VpreB and CD19 expression resistant to the inhibition following pre-BCR stimulation. Thus, Ca2+ down-regulates SLC and CD19 gene expression upon pre-BCR stimulation through inhibition of E2A by Ca2+/CaM. A general negative feedback regulation of the pre-BCR proteins as well as many co-receptors and proteins in signal pathways from the receptor was also shown. After the ordered recombination of Ig heavy chain gene segments, also Ig light chain gene segments are recombined together to create antibody diversity. The recombinations are orchestrated by the recombination activating gene (RAG) enzymes, other enzymes that cleave/mutate/assemble DNA of the Ig loci, and the transcription factor Pax5. A key feature of the immune system is the concept that one lymphocyte has only one antigen specificity that can be selected for or against. This requires that only one of the alleles of genes for Ig chains is made functional. The mechanism of this allelic exclusion has however been an enigma. Here pre-BCR signaling was shown to down-regulate several components of the recombination machinery including RAG1 and RAG2 through CaM inhibition of E2A. Furthermore, E2A, Pax5 and the RAGs were shown to be in a complex bound to key sequences on the IgH gene before pre-BCR stimulation and instead bound to CaM after this stimulation. Thus, the recombination complex is directly released through CaM inhibition of E2A. Upon encountering antigens, B cells must adapt to produce a highly specific and potent antibody response. Somatic hypermutation (SH), which introduces point mutations in the variable regions of Ig genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both SH and CSR. The AID enzyme has to be tightly controlled as it is a powerful mutagen. BCR signaling, which signals that good antibody affinity has been reached, was shown to inhibit AID gene expression through CaM inhibition of E2A.  SH increases the antigen binding strength by many orders of magnitude. Each round of SH leads to one or a few mutations, followed by selection for increased affinity. Thus, BCR signaling has to enable selection for successive improvements in antibodies (Ab) over an extremely broad range of affinities. Here the BCR is shown to be subject to general negative feedback regulation of the receptor proteins as well as many co-receptors and proteins in signal pathways from the receptor. Thus, the BCR can down-regulate itself to enable sensitive detection of successive improvements in antigen affinity. Furthermore, the feedback inhibition of the BCR signalosome and most of its protein, and most other gene regulations by BCR stimulation, is through inhibition of E2A by Ca2+/CaM. Differentiation to Ab-secreting plasmablasts and plasma cells is antigen-driven. The interaction of antigen with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. Genome-wide analysis showed that differentiation of B cells to Ab-secreting cell is induced by BCR stimulation through very fast regulatory events, and induction of IRF-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1 and Spi-B gene expressions were identified as immediate early events. Ca2+ signaling through CaM inhibition of E2A was essential for these rapid down-regulations of immediate early genes after BCR stimulation in initiation of plasma cell differentiation.

Differentes études ont montré que la sensibilité au Ca2+ du canal KCa3.1, un canal potassique indépendant du voltage, était conférée par la protéine calmoduline (CaM) liée de façon constitutive au canal. Cette liaison impliquerait la région C-lobe de la CaM et un domaine de $\ikca$ directement relié au segment transmembranaire S6 du canal. La CaM pourrait égalment se lier au canal de façon Ca2+ dépendante via une interaction entre un domaine de KCa3.1 du C-terminal (CaMBD2) et la région N-lobe de la CaM. Une étude fut entreprise afin de déterminer la nature des résidus responsables de la liaison entre le domaine CaMBD2 de KCa3.1 et la région N-lobe de la CaM et leur rôle dans le processus d'ouverture du canal par le Ca2+. Une structure 3D du complexe KCa3.1/CaM a d'abord été générée par modélisation par homologie avec le logiciel MODELLER en utilisant comme référence la structure cristalline du complexe SK2.2/CaM (PDB: 1G4Y). Le modèle ainsi obtenu de KCa3.1 plus CaM prévoit que le segment L361-S372 dans KCa3.1 devrait être responsable de la liaison dépendante du Ca2+ du canal avec la région N-lobe de la CaM via les résidus L361 et Q364 de KCa3.1 et E45, E47 et D50 de la CaM. Pour tester ce modèle, les résidus dans le segment L361-S372 ont été mutés en Cys et l'action du MTSET+ (chargé positivement) et MTSACE (neutre) a été mesurée sur l'activité du canal. Des enregistrements en patch clamp en configuration ``inside-out`` ont montré que la liaison du réactif chargé MTSET+ au le mutant Q364C entraîne une forte augmentation du courant, un effet non observé avec le MTSACE. De plus les mutations E45A et E47A dans la CaM, ont empêché l'augmentation du courant initié par MTSET+ sur le mutant Q364C. Une analyse en canal unitaire a confirmé que la liaison MTSET+ à Q364C cause une augmentation de la probabilité d'ouverture de KCa3.1 par une déstabilisation de l'état fermé du canal. Nous concluons que nos résultats sont compatibles avec la formation de liaisons ioniques entre les complexes chargés positivement Cys-MTSET+ à la position 364 de KCa3.1 et les résidus chargés négativement E45 et E47 dans la CaM. Ces données confirment qu'une stabilisation électrostatique des interactions CaM/KCa3.1 peut conduire à une augmentation de la probabilité d'ouverture du canal en conditions de concentrations saturantes de Ca2+. / The Ca2+ sensitivity of the voltage-insensitive calcium activated potassium channel of intermediate conductance KCa3.1 is conferred by calmodulin (CaM) constitutively bound to the membrane-proximal region of the channel intracellular C-terminus. A study was performed to investigate the nature of the residues involved in the CaM/KCa3.1 interactions and determine how these interactions could modulate the channel gating properties. A 3D-structure of the KCa3.1/CaM complex was first generated by homology modeling with MODELLER using as template the crystal structure of SK2.2/CaM complex (PDB: 1G4Y). The resulting structural model of KCa3.1 plus CaM predicts that the segment L361-S372 in KCa3.1 should be responsible for the Ca2+-dependent binding of the channel to the CaM-N lobe, with residues L361 and Q364 facing residues E45, E47 and D50 of CaM. To test this model residues in L361-S372 segment were substituted by Cys and the action of MTSET+ (positive charged) and MTSACE (neutral charged) measured on channel activity. Inside-out patch clamp recordings showed that the binding of the charged MTSET+ reagent to the Q364C mutant resulted in a strong current increase, an effect not seen with the neutral MTSACE. The mutations E45A and E47A in CaM prevented the current increase initiated by MTSET+ on the Q364C mutant. A single channel analysis confirmed that the binding of MTSET+ to Q364C caused an increase in the channel open probability by a destabilization of the channel closed state. Altogether, our results are compatible with the formation of ionic bonds between the positively charged Cys-MTSET+ complex at position 364 in KCa3.1 and the negatively charged E45 and E47 residues in CaM, and confirm that an electrostatic stabilization of the CaM/KCa3.1 interactions can lead to an increase in the channel open probability at saturating Ca2+.

Calmodulin (CaM) is a ubiquitous calcium-binding protein responsible for the binding and activation of a vast number of enzymes and signaling pathways. It contains two lobes that bind two calcium ions each, separated by a flexible central linker. This structural flexibility allows CaM to bind and regulate a large number of diverse protein targets within the cell in response to Ca2+ gradients. Voltage gated calcium channels (CaVs), as main sources of extracellular Ca2+, are crucial for a number of physiological processes, from muscle contraction to neurotransmission and endocrine function. These large transmembrane proteins open in response to membrane depolarization and allow gated entry of Ca2+ ions into the cytoplasm. Their regulation is currently the subject of intense investigation due to its pharmacological and scientific importance. CaM has been previously shown to pre-associate and act as a potent inhibitor of one class of high-voltage activated (HVA) channels called L-type channels via its interaction with their C-terminal cytoplasmic region. This interaction is primarily mediated by a conserved CaM-binding motif called the ‘IQ’ motif (for conserved isoleucine and glutamine residues), although the exact molecular details of its involvement in inactivation are currently unclear. Elucidation of these details was the primary objective of this dissertation. Recently, a novel sequence motif within this channel called ‘NSCaTE’ (N-terminal spatial calcium transforming element) has been described as an important contributor to calcium-dependent inactivation (CDI) of L-type channels. It was presumed to be unique to vertebrates, but we also show its conservation in a distantly related L-type channel homolog of Lymnaea stagnalis (pond snail). The interaction of CaM with a number of peptides representing the different regulatory motifs (IQ and NSCaTE) for both mammalian and snail isoforms was characterized in an attempt to better understand their role in CDI. Biophysical work with peptides as well as electrophysiology recordings with an N-terminal truncation mutant of Lymnaea CaV1 homolog were performed to expand our understanding of how the interplay between these channel elements might occur. In brief, the most striking feature of the interaction concerns the strong evidence for a CaM-mediated bridge between the N- and C-terminal elements of L-type channels. Further investigation of the CaM interaction with both IQ and NSCaTE peptides using Ca2+-deficient CaM mutants reveals a preference of both peptides for the Ca2+-C-lobe of CaM, and a much higher affinity of CaM for the IQ peptide, suggesting that the N-lobe of CaM is the main interaction responsible for the physiological effects of NSCaTE. These results are consistent with our electrophysiology findings that reveal a distinct buffer-sensitive CDI in wild type LCaV1 that can be abolished by the N-terminal truncation spanning the NSCaTE region. In addition to L-type channels, CaM has also been shown to have an indirect role in the regulation of low-voltage activated (LVA) or T-type channels (CaV3.x), via their phosphorylation by CaM-dependent protein kinase II (CaMKII). Using a primary sequence scanning algorithm, a CaM-binding site was predicted in a cytoplasmic region of these channels that was also previously shown to be important in channel gating. Biophysical experiments with synthetic peptides spanning this gating brake region from the three human and the single Lymnaea isoform strongly suggest that there is a novel, bona fide CaM interaction in this channel region, and also hint that this interaction may be a Ca2+-dependent switch of some sort. The results confirm a possible new role for CaM in the direct regulation of these channels, although the exact mechanism remains to be elucidated.

Protein interactions are the basis of synaptic function, and studying these interactions on a molecular level is crucial for understanding basic brain function, as well as mechanisms underlying neurological disorders. In this thesis, kinetic and mechanistic characterization of synaptic protein interactions was performed by using surface plasmon resonance biosensor technology. Fragment library screening against the reverse transcriptase of HIV was included, as it served as an outlook for future drug discovery against ligand-gated ion channels. The protein-protein interaction studies of postsynaptic Ca2+ -binding proteins revealed caldendrin as a novel binding partner of AKAP79. Caldendrin and calmodulin bind and compete at similar binding sites but their interactions display different mechanisms and kinetics. In contrast to calmodulin, caldendrin binds to AKAP79 both in the presence and absence of Ca2+ suggesting distinct in vivo functional properties of caldendrin and calmodulin. Homo-oligomeric β3 GABAA receptors, although not yet identified in vivo, are candidates for a histamine-gated ion channel in the brain. To aid the identification of the receptor, 51 histaminergic ligands were screened and a unique pharmacology was determined. A further requirement for identifying β3 receptors in the brain, is the availability of specific high-affinity ligands. The developed biosensor assay displayed sufficient sensitivity and throughput for screening for such ligands, as well as for being employed for fragment-based drug discovery. AMPA receptors are excitatory ligand-gated ion channels, involved in synaptic plasticity, and modulated by auxiliary proteins. Previous results have indicated that Noelin1, a secreted glycoprotein, interacts with the AMPA receptor. By using biochemical methods, it was shown that Noelin1 interacts directly with the receptor. The kinetics of the interaction were estimated by biosensor analysis, thereby confirming the interaction and suggesting low nanomolar affinity. The results provide a basis for functional characterization of a novel AMPA receptor protein interaction. The results demonstrate how secrets of synaptic protein interactions and function were revealed by using a molecular based approach. Improving the understanding of such interactions is valuable for basic neuroscience. At the same time, the technical advancements that were achieved to study interactions of ligand-gated ion channels by surface plasmon resonance technology, provide an important tool for discovery of novel therapeutics against these important drug targets.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.

The chicken hepatoma cell line, LMH, was evaluated with respect to its usefulness for studies of the regulation of heme metabolism. Levels of δ-aminolevulinate synthase mRNA arid accumulation of porphyrins were used to evaluate the heme biosynthetic pathway. Regulation of heme oxygenase-1 by known inducers was used as a measure of heme degradation. The induction of heme oxygenase-1 by sodium arsenite was characterized. AP-1 transcription factor elements and MAP kinase signal transduction pathways that modulate expression of endogenous heme oxygenase-1 and transfected heme oxygenase-1 reporter gene constructs in response to arsenite were delineated. In initial studies, the drug glutethimide was used alone or in combination with ferric nitrilotriacetate to induce δ-aminolevulinate synthase mRNA. Levels of porphyrins, intermediates in the heme biosynthetic pathway, and levels of δ-aminolevulinate synthase mRNA were increased by these treatments in a manner similar to those previously observed in the widely used model system, primary chick embryo liver cells. The iron chelator, deferoxamine, gave a characteristic shift in the glutethimide induced porphyrin accumulation in primary hepatocytes, but was found to have no, effect on LMH cells. Heme mediated repression of δ-aminolevulinate synthase mRNA levels was similar among primary hepatocytes and LMH cells. Heme oxygenase-1 was regulated by heme, metals, heat shock, and oxidative stress-inducing chemicals in LMH cells. Heat shock induction of heme oxygenase-1 mRNA levels was observed for the first time in primary chick embryo liver cells. These data supported the further use of LMH cells to elucidate mechanisms responsible for modulating heme oxygenase-1 gene expression in response to inducers. The remainder of the studies focused on the role of heme oxygenase-1 as a stress response protein. The oxidative stress inducer, sodium arsenite was used to probe the cellular mechanisms that control the expression of heme oxygenase-1. A series of promoter-reporter constructs were used to search the heme oxygenase-1 promoter for arsenite responsive elements. Several activator protein-1 (AP-1) transcription factor binding elements were identified by computer sequence analysis. Three of these sites, located at -1578, -3656, and -4597 base pairs upstream of the transcription start site, were mutated. The arsenite responsiveness of the reporter constructs containing mutated AP-1 elements was less than that of the same constructs containing wild type AP-1 elements. At least part of the arsenite-mediated induction of heme oxygenase-1 required the activity of AP-1 transcriptional elements. The MAP kinase signal transduction pathways and heme oxygenase-1 are activated by similar stimuli, including cellular stress. MAP kinases have been shown to exert control over gene expression through effects on the AP-1 family of transcription factors. The MAP kinases ERK, JNK, and p38 were activated by arsenite in LMH cells. Constitutively activated components of the ERK and p38 pathways increased expression of heme oxygenase-1 promoter-luciferase reporter constructs. Arsenite-mediated induction of heme oxygenase-1 was blocked by dominant negative ERK or p38 pathway components, and by specific inhibitors of MEK (upstream ERK kinase) or p38. In contrast, reporter gene expression was unchanged in the presence of constitutively activated JNK pathway components. Dominant negative JNK pathway components had no effect on arsenite induced heme oxygenase-1 gene activity. In summary, LMH cells were characterized as a new model system for the study of heme metabolism. This cell line was then used to delineate promoter elements and signaling pathways involved in the arsenite responsiveness of heme oxygenase-1 gene expression. Three AP-1 transcription factor binding sites in the heme oxygenase-1 promoter region were required for responsiveness to arsenite. The MAP kinases ERK and p38 were shown to play an integral role in arsenite-mediated induction of heme oxygenase-1. These studies elucidate one facet of heme oxygenase-1 regulation, and provide tools that will be useful in delineating additional regulatory mechanisms.

Orientadores: Antonio Carlos Boschiero, Luiz Fernando de Rezende / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T14:18:46Z (GMT). No. of bitstreams: 1 Santos_GustavoJorge_D.pdf: 3129731 bytes, checksum: b00bd77f6b06be14f135a76b6977ca47 (MD5) Previous issue date: 2014 / Resumo: Introdução: A Cálcio-Calmodulina quinase II (CaMKII) atua tanto na regulação da secreção de insulina com de neurotransmissores pela mesma via de sinalização. Além disso, a CaMKII é conhecida por ser a "molécula da memória", pois sua atividade é fundamental em sua formação. Portanto, hipotetizamos que células ß pancreática tem a capacidade de adquirir e estocar informações contidas em pulsos de cálcio, formando uma memória metabólica. Métodos: Para comprovar nossa hipótese, desenvolvemos um novo paradigma de exposição de células ? a pulsos de 30 mM de glicose, seguido de uma período de consolidação (24 hrs) para excluir qualquer efeito agudo do metabolismo da glicose. Após esse período analizamos a secreção de insulina (RIA), expressão proteica (Western blot), a resposta secretória frente a uma "rampa de glicose" e o Ca2+ citoplasmático induzido por glicose. Resultados: Células ß expostas a pulsos de glicose (30 mM) mostraram maior secreção de insulina estimulada por glucose, evidenciando a memória metabólica a qual foi totalmente dependente a CaMKII. Esse fenômeno foi refletido na expressão proteica de proteínas importantes na sinalização do cálcio e na secreção de insulina. Além disso, células expostas ao regime de pulsos de glucose apresentaram maior expressão do MAFA, um fator de transcrição chave para a função da célula ß. Conclusão: Em suma, assim como neurônios, células ß tumorais (MIN6), ilhotas de camundongos e de humanos são capazes de adquirir, estocar e evocar informações / Abstract: Backgroun: Ca2+/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Memory is the ability to acquire, to store and to evocate any kind of information. In CNS, the process behind this phenomenon in the Long-Term Potentiation (LTP) and is known that it requires Ca2+ to occur. In additional, CaMKII is necessary to store information during LTP. In pancreatic ß-cells, CaMKII plays pivotal role during GSIS process. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in Ca2+ pulses as a form of "metabolic memory", just as neurons store cognitive information. Methods: To test this hypothesis, we developed a novel paradigm of pulsed exposure of mice and human ß-cells to intervals of high glucose, followed by a 24-hour consolidation period to eliminate any acute metabolic effects. After this period, we analyzed insulin secretion (by RIA), protein expression (by Western blot), response to a glucose-ramp and the glucose-induced Ca2+ influx. Results: Strikingly, ß-cells exposed to this high-glucose pulse paradigm exhibited significantly stronger insulin secretion. This metabolic memory was entirely dependent on CaMKII. We also observed, in pulse group, an increase in Ca2+ influx induced by glucose. In additional, metabolic memory was reflected on the protein level by increased expression of proteins involved in GSIS and Ca2+-dependent vesicle secretion, such as GCK, Cav1.2, SNAP25, pCaMKII and pSynapsin. Finally, we observed in human islet elevated levels of the key ß cell transcription factor MAFA. Discussion: Based on or findings we conclude that pancreatic ß cells, either from mice or humans, have the ability to acquire, store and retrieve information. This process is CaMKII-dependent and is due to modifications in the glucose-sensing machinery of the cell, since we observed an increase in GSIS and Ca2+ influx together with an increase in several proteins involved in this process. Our findings suggests that MAFA is the key effector in this memory, since (a) it is a potent activator of insulin gene, (b)is activated by CaMKII and (c) its expression is increased even 24 hours after the last pulse. Conclusion: In summary, like neurons, human and mouse ß-cells are able to acquire and retrieve information / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular