Teses / dissertações sobre o tema "Ca(2+)-calmodulin dependent protein kinase"

Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第10449号
医博第2648号
新制||医||844(附属図書館)
UT51-2003-T275
京都大学大学院医学研究科脳統御医科学系専攻
(主査)教授 月田 承一郎, 教授 中西 重忠, 教授 武藤 誠, 教授 成宮 周
学位規則第4条第1項該当

Thesis (Ph. D.)--Medical College of Ohio, 2003.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Howard Rosenberg. Document formatted into pages: iv, 144 p. Title from title page of PDF document. Includes bibliographical references (p. 104-132).

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.

Epithelial to mesenchymal transition (EMT) is an important process in embryonic development, tissue repair, inflammation, and cancer. During EMT, epithelial cells disassemble cell-cell adhesions, lose apicobasal polarity, and initiate migratory and invasive processes that allow individual cells to colonize distant sites. It is the means by which non-invasive tumors progress into malignant, metastatic carcinomas. In vitro, EMT occurs in two steps. First, cells spread out, increasing in surface area and pushing the colony borders out. Then cells contract, pulling away from neighboring cells and rupturing cell-cell junctions, resulting in individual highly migratory cells. Recent discoveries indicate that calcium signaling is central in EMT. Both previous data with patch clamping and new calcium imaging data show a series of calcium influxes in cells induced to undergo EMT with hepatocyte growth factor (HGF). It has also been shown that blocking calcium signaling prevents EMT from progressing normally. However, it is not known if calcium alone is sufficient to drive EMT behaviors. By experimentally triggering calcium influxes with an optigenetic cation channel, the behaviors that calcium influxes induce can be determined noninvasively, without use of drugs that may have secondary effects. The results of using the optigenetic set up along with live cell imaging are that cells become more motile and disrupt normal epithelial cell-cell adhesions. This behavior is believed to be due to increased cell contractility downstream of calcium signaling, and is dependent on Ca2+/calmodulin-dependent protein kinase II (CaMKII). When cells are pre-treated with CaMKII inhibitor before HGF addition, they undergo the spreading step of EMT without subsequent cellular contraction and rupture of cell-cell junctions. CaMKII is a protein kinase that is activated by binding Ca2+/calmodulin, and is a known downstream component of calcium signaling. CaMKII is known to affect the actin cytoskeleton by both physically bundling actin filaments to increase their rigidity, and through signaling by activation of myosin light chain kinase (MLCK), which has a role in stress fiber formation. Immunofluorescence did not show colocalization of CaMKII with actin, ruling out regulation through actin bundling. However, CaMKII does appear to have a role in stress fiber formation. EMT induced with HGF treatment results in increased numbers of stress fibers as well as trans-cellular actin network formation, both actin structures decorated with non-muscle myosin II (NMII). CaMKII inhibition not only blocks these actin formations, but it also decreases stress fiber levels below basal unstimulated levels in cells that have not been treated with HGF. This suggests that CaMKII has a role in regulating contractility through cellular actin networks, indicating a mechanism for calcium's role in cellular contractility in EMT.

Cytosolic calcium (Ca2+) levels are critical for the control of muscle contraction and are tightly regulated by a variety of Ca 2+ transport systems localized in various membranes. Ca2+ binding proteins such as calmodulin (CaM) and Ca2+/CaM-dependent protein kinases (CaM Kinases) are believed to exert major regulatory control on Ca2+ activity. Previous studies in this lab led to the cloning of a cDNA encoding a CaM Kinase II beta isoform from skeletal muscle that differed from the classical beta isoform by the inclusion of three alternatively spliced exons in the variable domain which were enriched in proline residues. A CaM Kinase, assumed to be localized in the sarcoplasmic reticulum (SR), has been implicated in the regulation of excitation-contraction (E-C) coupling. We hypothesized that this novel CaM Kinase II beta isoform called SOCK (Son Of CaM Kinase) may be the CaM Kinase II isoform that regulates E-C coupling by being targeted to specific regions of the SR, whereby it phosphorylates critical Ca2+ transporting proteins such as the ryanodine (RyR) and dihydropyridine (DHPR) receptors in response to changes in Ca2+ levels. (Abstract shortened by UMI.)

Expression levels of GABAA receptor subunits and activity of the Ca++ /calmodulin-dependent protein kinase II (CaM kinase II) enzyme system were evaluated in an in vitro hippocampal neuronal culture model of epilepsy. Treatment of hippocampal neuronal cultures with Mg++-free media for 3 hours results in the induction of an enduring "epileptic" state as evidenced by the expression of spontaneous recurrent seizure (SRS) discharge. The induction of the SRS activity was shown to be a N-methyl-D-aspartate (NMDA) receptor, Ca++-dependent mechanism (Sombati and DeLorenzo, J Neurophys., 73 (4), 1995). Significant and long-lasting decreases in mRNA expression for the GABAA α2 and α5 receptor subunits were observed in association with the induction of SRSs in this model, while levels for α1, β2 and γ2 subunits showed no significant change. Irreversible [3H]-flunitrazepam saturation binding analysis in membrane preparations demonstrated a significant decrease in specific binding in association with the SRS activity observed in this model. No changes in GABAA P subunit immunoreactivity were detected. Selective suppression of the GABAA α2 subunit protein levels in hippocampal neuronal cultures using antisense oligonucleotide technology caused a significant decrease in the amplitude of spontaneous inhibitory postsynaptic currents (sIPSC). CaM kinase II is highly enriched in the brain and mediates many processes essential to neuronal function and viability. Induction of SRSs in hippocampal cultures were associated with a long-lasting and significant decrease in activity of CaM kinase II. Addition of 2-amino-5-phosphovaleric acid to the low Mg++ treatment solution prevented the decrease in CaM kinase II activity. Suppression of CaM kinase II activity in hippocampal cultures by treatment with either an antisense oligonucleotide specific for a CaM kinase II or KN93 (selective CaM kinase II inhibitor) resulted in significant reductions in IPSC amplitude. This data suggests that CaM kinase II can act to regulate GABAergic inhibitory function in hippocampal cultures. The findings of this study demonstrate long-lasting decreases in GABAA receptor expression and activity of CaM kinase II, which may contribute to the induction of the "epileptic" state of this model.

La polykystose rénale autosomique dominante (ADPKD) est la maladie monogéniquehumaine la plus fréquente (prévalence 1/800). Les gènes responsables de cette maladie sont PKD1(codant pour PC1) ou PKD2 (codant pour PC2). La maladie évolue vers l’insuffisance rénale terminale.Aujourd’hui, seul un traitement symptomatique est proposé aux malades. Les mécanismes à l’originede l’ADPKD sont mal connus. Les modèles animaux permettent de mieux comprendre laphysiopathologie d’une maladie. Il n’existe pas de bon modèle de polykystose (même causemoléculaire, même mode de transmission, même signes cliniques). En utilisant la transgénose degrands fragments, nous avons créé un modèle de surexpression de PKD2 humain. Le transgène estsous le contrôle de son promoteur naturel humain. Cette souris exprime deux fois plus de PC2 queles sauvages. Elle ne présente que quelques microkystes mais une tubulopathie associant défaut deconcentration des urines et protéinurie tubulaire. La surexpression de PC2 inhibe l’expression degènes codant pour des protéines de la matrice extracellulaire. Le phénotype cellulaire de cesanimaux est remarquable : un tiers des cellules présentent un nombre élevé de centrosomes. Cephénotype cellulaire a été retrouvé chez des souris sous exprimant Pkd2 et chez des souris sousexprimant Pkd1. Ce caractère multicentrosomique est corrigé en incubant les cellules avec uninhibiteur de CaMKII ou en croisant nos souris transgéniques avec une souris KO de Camk2. Nousavons réussi à lier CaMKII, la duplication des centrosomes et les polycystines, in vitro et in vivo. Ceciamène un éclairage nouveau sur la duplication du centrosome et la physiopathologie de l’ADPKD
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common monogenic human disease (prevalence 1/800). Genes responsible for this disease are PKD1 (encoding PC1) or PKD2 (encoding PC2). The disease progresses to end stage renal disease. Today, only symptomatic treatment is offered to patients. The mechanisms underlying the ADPKD are unknown. Animals models allow better understand the disease’s pathophysiology. There is no good model of ADPKD (same molecular cause, same clinical signs). We created a mice model of human PKD2 overexpression. The transgène is under the control of its human natural promoter. This mouse expresses PC2 twice as much as the wild. It shows only few microcysts but tubulopathy involving lack of urine concentration and tubular proteinuria. PC2 overexpression inhibits the expression of genes encoding proteins of the extracellular matrix. The cellular phenotype of these animals is special : one third of the cells have a high number of centrosomes. This cellular phenotype was found in Pkd2 Knockout mice and in Pkd1 knockout mice. This multicentrosomic character is corrected by incubating the cells with a CaMKII inhibitor or by crossing our transgenic mice with Camk2 knockout mice. We propose a link between CaMKII, Centrosome duplication and polycystin in vitro and in vivo. This brings a new light on centrosome duplication and pathophysiology of ADPKD

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

La potentialisation à long terme (LTP) est une forme de plasticité synaptique caractérisée par une augmentation durable de l'efficacité synaptique suite à une stimulation tétanique des fibres afférentes. Son maintien se trouve altéré au cours du vieillissement. Le but de ce travail a été d'analyser au niveau transcriptionnelles mécanismes de la LTP au sein du gyrus denté et leurs altérations au cours du vieillissement. Ce travail a été réalisé grâce au développement de nouvelles méthodes d'analyse quantitative, plus sensibles et mieux adaptées aux questions physiologiques posées. Deux d'entre elles portent sur l'analyse de l'expression des ARNm au niveau cellulaire : (i) la reconstruction tridimensionnelle des niveaux d'expression de plusieurs ARNm dans une structure et (ii) le double marquage radioactif en hybridation in situ permettant une analyse comparative, quantitative et simultanée de deux ARNm sur une coupe de tissu. Les analyses de l'expression des ARNm (Zif268, Homer, syntaxine lB, CaMKII, APP, a-synucléine) dans le gyrus denté après induction de la LTP révèlent la diversité de la réponse transcriptionnelle au sein même de la structure au niveau cellulaire mais également au niveau subcellulaire chez le jeune rat adulte et au cours du vieillissement. Les résultats montrent une expression spatio­temporelle hétérogène pour Zif268, Homer et syntaxine lB dans le gyrus denté des jeunes rats adultes et une altération de la transmission transsynaptique de la LTP au cours du vieillissement, accompagnée de modifications dans l'expression des ARNm AP [KPI-], CaMKII et a-synucléine qui pourraient expliquer en partie l'absence de maintien de la LTP dans le gyrus denté. En parallèle, afin de permettre une analyse à grande échelle des profils d'expression des ARNm dans le cadre de la LTP, une approche de puces à ADN utilisant deux radioéléments différents a été développée et offre la sensibilité et la reproductibilité nécessaires à une analyse fine comme celle de la LTP
Long-term potentiation (LTP), a model for synaptic plasticity, is characterised by a persistent increase in synaptic strength following tetanie stimulation of afferent fibres. Its maintenance is altered during ageing. The aim of the present work was to analyse, at a transcriptional level, first LTP mechanisms within the dentate gyrus and second changes in LTP during ageing. The work was performed using novel methods of quantitative analysis. These methods are more sensitive and best suited to address the physiological questions. Two of them rely on the analysis of messenger RNA expression at a cellular level: (i) the tridimensional reconstruction of expression levels of several mRNA within a structure and (ii) use of double radioactive labelling in in situ hybridisation allowing a comparative, quantitative and simultaneous analysis of two mRNA in a tissue section. The analyses of mRNA expression (Zif268, Homer, syntaxin lB, CaMKII, APP and a-synuclein) in the dentate gyrus following LTP induction, revealed the diversity in the transcriptional response within the structure both at the cellular and subcellular levels in young adult rats and during ageing. The results showed: (i) a heterogeneous spatio-temporal expression for Zif268, Homer et syntaxin lB in the dentate gyrus of young adult rats; (ii) an alteration of the transsynaptic transmission of LTP during ageing, and (iii) alterations of mRNA expression for APP[KPI-], CaMKII and a­synuclein during ageing. These last results can potentially explain the lack of LTP maintenance in the dentate gyrus. In parallel, to allow a large-scale analysis of mRNA expression profiles in LTP, a microarray approach using two different radioelements was developped. This approach provided the sensitivity and the reproducibility that are required for the fine analysis of a physiological event such as LTP

L’évolution des populations occidentales s’accompagne d’une augmentation de la sédentarité et des maladies métaboliques qui accroissent les problèmes de santé. Ces évolutions ont des répercussions sur le muscle squelettique qui voit sa capacité à produire de l’énergie aérobie diminuer. Néanmoins, le muscle squelettique est très plastique et les capacités oxydatives musculaires s’améliorent rapidement par l'activité physique. Les mitochondries sont des éléments majeurs des capacités oxydatives musculaires et la compréhension des mécanismes moléculaires qui régissent la biogenèse et la fonction mitochondriale est nécessaire pour prescrire au mieux l’activité physique.L’exercice intermittent semble être de plus en plus utilisé dans la pratique. Plusieurs arguments sont mis en avant pour préconiser cette modalité : 1) le temps passé à haute consommation d’oxygène, 2) la haute intensité et 3) les perturbations métaboliques induites par les variations d’intensité au cours de l’exercice. Cependant, l’influence des perturbations métaboliques sur les capacités oxydatives musculaires n’a pas encore été clairement démontrée. L’objet des mes travaux de thèse s’est donc focalisé sur ces perturbations métaboliques et leurs effets sur les voies de signalisation impliquées dans la biogenèse mitochondriale. Afin de caractériser l’implication des perturbations métaboliques dans la stimulation des voies de signalisation de la biogenèse mitochondriale, nous avons comparé l’influence d’exercices aigus sur ces voies de signalisation. Deux protocoles nous ont permis d’investiguer l’influence des variations métaboliques. Le premier a consisté, lors d’un exercice de intermittent, à identifier la durée du cycle induisant les plus grandes perturbations métaboliques et à caractériser les effets de la modalité d’exercice sur un exercice de 30 minutes de pédalage à 70%WRpic. Le second protocole visait à déterminer l’influence de la répétition des perturbations métaboliques sur les voies de signalisation régulant la biogenèse mitochondriale.Afin d’identifier la durée de cycle produisant le plus de variations métaboliques, nous avons analysé l’évolution de la consommation d’oxygène et quantifié les variations métaboliques. Pour cela nous avons utilisé trois paramètres : 1) un paramètre quantitatif, 2) un paramètre qualitatif et 3) un index associant les paramètres quantitatif et qualitatif. La comparaison de trois durées de cycle différentes (30s d’effort:30s de récupération passive ; 60s:60s et 120s:120s) nous a permis de mettre en évidence que la modalité 60s:60s est celle qui induit le plus de variations métaboliques et cela pour une dépense énergétique identique pour les trois modalités.Notre seconde étude a consisté à comparer 30 minutes de pédalage à 70%WRpic sous deux modalités différentes : continue (1 bloc de 30min) et intermittente (30 bloc de 1min entrecoupés de 1min). La répétition de phase d’exercice et de repos lors de l’exercice intermittent créée plus de perturbation du métabolisme et entraîne une phosphorylation supérieure de l'AMPK, CaMKII et p38 MAPK. Ces kinases sont situées en amont de PGC-1α, un important régulateur de la biogenèse mitochondriale dans le muscle squelettique. Ces résultats mettent donc en évidence un effet spécifique des perturbations métaboliques sur les voies de signalisation contrôlant la biogenèse mitochondriale.Ces travaux ouvrent de nouvelles perspectives sur les méthodes de réentraînement de personnes sédentaires ou atteintes de pathologie chronique. Les futurs travaux viseront à confirmer nos résultats lors d’interventions chroniques et d’explorer ces effets chez différentes populations
Western life evolution is associated with an increase in sedentary behaviours and metabolic diseases leading to health alteration. This evolution affects the skeletal muscle, which is characterized by a decrease in its ability to produce aerobic energy. However, skeletal muscle is a highly malleable tissue, capable of considerable metabolic adaptations in response to physical activity. Mitochondria produce the aerobic energy within the skeletal muscle. Understanding the molecular mechanisms that regulate mitochondrial biogenesis and its function is necessary to improve physical activity prescription.The intermittent exercise is currently used in rehabilitation programs. Several arguments are put forward to utilizing this method: 1) the time spent at high oxygen consumption, 2) the high intensity of exercise and 3) the metabolic disturbances induced by variations of intensity during exercise. However, the influence of metabolic disturbances on muscle oxidative capacity has not been clearly demonstrated. The purpose of my thesis work has therefore focused on these metabolic perturbations and their effects on signalling pathways involved in mitochondrial biogenesis. In order to characterize the influence of metabolic disturbances on the signalling pathways involved in mitochondrial biogenesis, we compared the influence of acute exercises. We realized two protocols to investigate the influence of metabolic disturbances. The first study compared three intermittent exercises in order to identify the optimal duty-cycle duration to induce the biggest metabolic disturbances and to compare metabolic responses of intermittent and continuous exercise performed at 70%WRpic. The second protocol evaluated the influence of the repetition of metabolic disturbances on signalling pathways involved in mitochondrial biogenesis.In order to identify the duty-cycle duration producing more metabolic fluctuations, we analysed the changes of oxygen consumption and quantified metabolic variations. We used three parameters: 1) a quantitative parameter, 2) a qualitative parameter, and 3) an index combining quantitative and qualitative parameters. Comparison of three different duty-cycle durations (30s work:30s passive recovery; 60s:60s, and 120s:120s) revealed that the 60s:60s modality induces more metabolic fluctuations for a same energy expenditure.Our second study compared 30 minutes of pedalling at 70%WRpic realized by two different modalities: continuous (30min 1 block) and intermittent (30 1min block interspersed by 1min of passive recovery). Repetition of transitions from rest to exercise during the intermittent exercise creates higher metabolic disturbances and leads to a higher phosphorylation of AMPK, p38 MAPK and CaMKII. These kinases are upstream of PGC-1α, an important regulator of mitochondrial biogenesis in skeletal muscle. All together, these results demonstrate that metabolic disturbances are involved in mitochondrial signalling pathways activation.This work opens up new perspectives on exercise training prescription for sedentary or chronic pathology people. Future work will aim to confirm our results in chronic interventions and explore these effects in different populations

A combination of biochemical, immunochemical, and molecular biological techniques have been employed to purify and characterize a rat brain Ca²⁺/calmodulin-dependent protein kinase. The enzyme, named type II Ca²⁺/calmodulin-dependent protein kinase (type II CaM kinase), was identified in rat brain homogenates by its ability to phosphorylate site II on the synaptic vesicle associated protein synapsin I.

Type II CaM kinase has been purified 290 fold over crude homogenates and is found to be composed of multiple copies of two different subunits. Both subunits copurify with kinase activity and are coprecipitated with kinase activity by an anti-kinase monoclonal antibody. The two subunits have molecular weights of 50,000 (α) and 58,000/60,000 (β), and are present in a 3:1 α:β ratio. The type II CaM kinase holoenzyme has a sedimentation coefficient of 16.4 S, a Stokes radius of 95 Å, and a calculated molecular weight of 650,000. A dodecameric holoenzyme consisting of 9 α subunits and 3 β subunits has been proposed. The purified type II CaM kinase phosphorylates several substrates, in addition to synapsin I, at a significant rate, and may therefore be responsible for a number of neuronal responses to Ca²⁺.

The α subunit of type II CaM kinase has a number of biochemical characteristics which are similar to the major protein component of a subcellular fraction which is derived from brain postsynaptic densities (PSDs). A direct comparison between the a subunit of type II CaM kinase and the major PSD protein using immunochemical and biochemical techniques has revealed that they are in fact very similar or identical proteins.

Two approaches have been taken to further characterize the subunits of type II CaM kinase at a molecular level. The first approach has been to isolate cDNA clones which code for the β subunit. A number of clones have been isolated and sequenced. The ammo acid sequence for the β subunit (predicted from the cDNA sequence) is homologous to several other protein kinases. Southern blot analysis with a β subunit cDNA indicates the existence of a type II CaM kinase multigene family. The second approach to the molecular characterization of the type II CaM kinase subunits has been to determine the amino acid sequence of peptides derived from the α subunit. Two regions of α subunit sequence have been determined, and both are found to be homologous to regions of β subunit amino acid sequence deduced from β subunit cDNA clones.

The molecular characterization of neuronal type II CaM kinase in vitro has both provided insight into the possible function of the enzyme in vivo and suggested experimental approaches which may eventually allow its in vivo function to be directly addressed.

Four monoclonal antibodies generated against the Type II CaM kinase have been characterized. Two of these antibodies were used to confirm that both alpha and beta subunits were part of the holoenzyme complex. I also developed liquid phase and solid phase radioimmunoassays for the kinase.

With the solid phase radioimmunoassay, the distribution of the kinase in rat brain was examined. This study revealed that the concentration of the kinase varies markedly in different brain regions. It is most highly concentrated in the telencephalon where it comprises approximately 2% of total hippocampal protein, 1.3% of cortical protein and 0.7% of striatal protein. It is less concentrated in lower brain regions ranging from 0.3% of hypothalamic protein to 0.1% of protein in the pons/medulla. The unusually high concentration of the kinase in telencephalic regions may confer upon their neurons specialized responses to calcium that are different from those of neurons in lower brain regions.

The association of the kinase with elements of the cytoskeleton was also investigated. The results of this study showed that autophosphorylation causes an increase in the association of the enzyme with taxol-polymerized microtubules and F-actin. This increase in association was reversed by dephosphorylating phosphokinase with protein phosphatase. These results suggest that autophosphorylation could constitute a mechanism for the regulation of the subcellular associations of the Type II CaM kinase by neuronal activity.

Calcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies.

We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.

In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.

Dissertation