Il a déjà été démontré que les statines (ou inhibiteurs de la HMG-CoA réductase) sont efficaces pour réduire le LDL-cholestérol et elles se sont depuis établies comme étant le pilier dans le traitement de la dyslipidémie. Toutefois, environ 10 pourcent des utilisateurs de statines souffrent d'effets indésirables, généralement sous forme de myopathie qui est souvent accompagnée d’un taux élevé de la créatine kinase (CK) plasmatique. Il est fréquent que les patients doivent arrêter les statines à cause d’un taux de CK dépassant un seuil de référence. Nous avons examiné le taux de CK de près de 6000 participants de la biobanque de l’ICM, qui ont récemment été génotypés à l'aide de la micropuce d'ADN ExomChip d'Illumina. Des études antérieures ont démontré une association significative entre le taux de CK plasmatique et des polymorphismes génétiques et nous avons cherché à répliquer ces résultats par association génétique et à l'aide du test SKAT pour les polymorphismes rares. Nous avons répliqué les résultats dans le gène CKM (rs11559024, p=1.59x10-23) et le gène LILRB5 (rs12975366, p=1.44x10-26) dans le chromosome 19. Nous espérons que ces résultats seront éventuellement utilisés en clinique pour la prédiction des taux de référence de CK personnalisés selon le profil génétique des patients utilisateurs de statines. / Statins (HMG-CoA reductase inhibitors) have been shown to reduce LDL-cholesterol and are undoubtedly the mainstay in the treatment of hyperlipidemia. Approximately 10 percent of statin users suffer from adverse side effects, the most common being muscle myopathy. Muscle myopathy is often accompanied by elevated levels of plasma creatine kinase (CK). Oftentimes, patients are taken off statins after their CK levels surpass a reference threshold. We looked at CK levels in the MHI Biobank, which have recently been genotyped in over 6000 participants with the Illumina ExomChip. Prior studies have found significant association between plasma CK levels and genetic variants and we aimed to replicate these findings using a genome wide association and a SKAT burden test for rare variants. We were able to replicate findings in the CKM gene (rs11559024, p=1.59x10-23) and LILRB5 gene (rs12975366, p=1.44x10-23) in chromosome 19. We hope that these results will eventually be utilized in clinical statin care by aiding in the prediction of personalized reference CK levels based on genetic information for patients using statins.

The Cardiac Profile, a pattern of serum enzyme changes seen within seventy-two hours after an AMI, is diagnostic aid for detecting occurrence of infarcts. The effects of exercise stress on the Cardiac Profile aid clinicians in avoiding diagnostic errors in patients immediately after exercise. Five male volunteers ran from six to ten miles. Serum enzyme levels were monitored serially three days before and five days after stress. Enzyme activity was determined spectrophotometrically and electrophoretically. Significant increases in total CPK and LDH were seen. An LDH 'one-two flip' occurred eight hours after exercise. No MB-CPK was found following the run.

As atividades enzimáticas e isoenzimáticas da CK e da LD foram determinadas nos tecidos gástricos neoplásico e da margem de ressecção e no soro de indivíduos com adenocarcinoma de estômago, submetidos à gastrectomia. O tecido gástrico neoplásico aapresentou índices CKBB/Cktotal e LD5/LD1, bem como atividade da LD5, superiores aos da margem de ressecção correspondente. No pré-operatório, os indivíduos estudados apresentaram elevação da atividade sérica da CK BB (100%) e da CK MB (69%). Este fato, possivelmente, resultou da liberação dessas isoenzimas pelo próprio neoplasma ou, também pelo tecido normal adjacente. As atividades séricas das isoenzimas da LD apresentaram-se dentro dos valores de referência, nesse período. Após a gastrectomia, houve aumento significativo das atividades isoenzimáticas séricas da CK e da LD relação àquelas do pré-operatório, notadamente, no 1º período pós-operatório. Tais alterações foram atribuídas à liberação dessas isoenzimas pelos tecidos lesados durante o ato cirúrgico. / Abstract not available

Estudos recentes têm associado à prática de exercício físico de alta intensidade e de forma inabitual a alterações musculares que incluem lesões estruturais das miofibras. Nessa perspectiva, a suplementação de aminoácidos de cadeia ramificada (BCAA) destaca-se como uma estratégia eficaz no controle do dano celular e na promoção da recuperação muscular. Uma vez que, dentre os BCAA, a leucina é o que apresenta maior influência sobre a regulação de mecanismos anabólicos que incluem o estímulo ao aumento da taxa de tradução de RNAm mediado pela proteína quinase mTOR, se especula quais os possíveis efeitos da sua suplementação isolada em um modelo de exercício físico extenuante e inabitual. Buscamos então, com o presente estudo, avaliar o efeito da suplementação com leucina sobre as concentrações séricas de creatina quinase - marcadores de lesão muscular - e sobre a expressão e fosforilação das proteínas mTOR, 4E-BP1, S6K1, IKK, IkB-α, NF-κB - componentes de vias envolvidas na síntese de proteínas e da inflamação - no músculo esquelético de ratos adultos não treinados, submetidos a uma sessão de exercício até a exaustão. Para tanto, 48 ratos machos adultos da linhagem Sprague-Dawley foram distribuídos em 10 grupos, sendo que T3L, T48L E T72L receberam suplementação com leucina [leucina (135 mg/100 g) e T3A, T48A e T72A receberam uma mistura de aminoácidos não essenciais (135 mg/100 g)] ao final de uma sessão de exercício físico agudo até a exaustão; T3E, T48E e T72E - não receberam suplementação e foram submetidos ao protocolo de exercício; o grupo controle não recebeu suplementação e não foi submetido ao protocolo de exercício. Em seguida os animais foram eutanasiados 3, 48 e 72 horas após o termino do exercício para análise dos marcadores investigados. Entre os parâmetros avaliados houve diferença significativa na atividade da creatina quinase entre os grupos TL3 e TL4. No entanto os marcadores biomoleculares não apontaram diferenças entre os grupos. No entanto a fosforilação do NF- Kb da fração nuclear demonstrou uma tendência a ser menor nos grupos suplementados com leucina e o mesmo acontece com a MCP-1 (proteína quimioatraente de monócitos). Visto isso, podemos concluir que, em nossas condições experimentais, as vias de sinalização estudadas estavam envolvidas na diminuição da atividade da creatina quinase, porém, nossos resultados apontam que a suplementação com leucina pode estar envolvida em mecanismos que atenuem o dano celular após o exercício agudo em ratos não treinados. / Recent studies have associated infrequent exercise of high intensity to muscle changes that include structural lesions in myofibres. The supplementation of branched chain aminoacids (BCAA) stands out as an effective strategy in controlling the cell damage and promoting muscle recovery. As leucine is the BCAA exerting more influence on the regulation of anabolic mechanisms that include stimulating the increase of mRNA translation rate mediated by the protein kinase mTOR, it is speculated the potencial effects of leucine supplementation alone in a model of strenuous and infrequent exercise. In the present study, we evaluate the effect of leucine supplementation on serum levels of creatine kinase - marker of muscle damage - and on the expression and phosphorylation of proteins mTOR, 4E-BP1, S6K1, IKK, IkB-α, NF-κB - components of pathways involved in protein synthesis and inflammation - in the skeletal muscle of untrained adult rats, after a single acute bout of exercise to exhaustion. Forty eight adult male Sprague-Dawley rats were divided into 10 groups: T3L, T48L and T72L groups were given leucine supplementation [leucine (135 mg/100 g), and T3A, T48A and T72A groups were given a mix of non-essential aminoacids (135 mg/100 g)] at the end of the acute bout of exercise to exhaustion; T3E, T48E and T72E groups did not receive supplementation and underwent the exercise protocol; control group was not given supplementation nor exercise protocol. The animals were killed 3, 48 and 72 hours after the end of exercise to analyze the markers under study. Among the parameters assessed, there was a significant difference in the activity of creatine kinase between groups TL3 and TL4. However, no differences were seen in the biomolecular markers between groups. Nevertheless, the nuclear fraction of NF- Kb phosphorylation tended to be lower in the groups supplemented with leucine and the same was seen with MCP-1 (monocyte chemoattractant protein). Taken together, we conclude that the signalling pathways studied, in our experimental condition, do not seem to be involved in the reduced activity of creatine kinase; conversely, our results indicate that leucine supplementation may be involved in mechanisms attenuating cellular damage after a single acute bout of exercise in untrained rats.

La bioénergétique moléculaire des systèmes est une nouvelle direction de recherche scientifique qui s'inscrit dans la Biologie des Systèmes. Elle étudie et décrit le métabolisme énergétique intégré cellulaire non seulement comme un réseau de réactions mais aussi décrit ses aspects spatiaux (organisation) et temporels (dynamique). La bioénergétique moléculaire des systèmes considère l'organisation spatiale intracellulaire comme un processus dynamique dont la topologie elle-même renferme des informations. Ce projet tend à mettre l'accent sur une approche expérimentale décrivant les réseaux de phospho-transferts intracellulaire. Le principal objectif de ce travail fut la description comparative de l'hétérogénéité de la compartimentation des nucléotides adényliques et la complexité structurale et fonctionnelle des communications entre la mitochondrie et d'autres structures ou processus intracellulaire (cytosquelette, glycolyse) dans les cellules nerveuses (synaptosomes) et cardiaques (cardiomyocytes adultes et lignée cancéreuse de cellules HL-1). Les résultats de ce projet ont démontré 1/ la régulation de la perméabilité de la membrane externe mitochondriale par le facteur X (association de la tubuline hétérodimérique avec la porine voltage dependent anion channel), 2/ le couplage fonctionnel entre la creatine kinase mitochondriale et l'adénine nucléotide translocase , 3/ les mécanismes de régulation de la respiration mitochondriale in vivo qui ont permis d'établir un schéma de l'Interactosome Mitochondrial, 4/ les variations de régulation métabolique associées au cancer et 5/ l'hétérogénéité de la diffusion des nucléotides adényliques et leur micro- voire nano-compartimentation après activation des créatines kinases (par spectroscopie à corrélation de fluorescence).

Une propriété clé des systèmes biologiques est la présence d'un réseau d'interactions protéiques, crucial pour toute fonction cellulaire comme par exemple la régulation du métabolisme énergétique. Deux enzymes clé impliquées dans cette régulation sont la créatine kinase (CK), dont la fonction consiste dans la gestion du stock et du transfert d'énergie, et la protéine kinase activée par l'AMP (AMPK), qui régule l'homéostasie énergétique au sein de la cellule et de l'organisme entier. Dans un premier temps un crible de double hybride en levure original fut appliquée afin d'identifier de nouveaux partenaires d'interaction de la CK cytosolique du cerveau (BCK) et de l'AMPK dans le cerveau humain. Différents candidats d'interaction furent identifiés, dont des protéines membranaires associées aux vésicules (VAMP) interagissant avec les deux kinases. L'interaction AMPK-VAMP fut confirmée par co-immunoprecipitation à partir de vésicules synaptiques, mais ne menait pas à la phosphorylation de VAMP, suggérant que VAMP recrute AMPK pour la régulation de processus d'endo- et d'exocytose. Une seconde stratégie combinant un essai d'interactions biophysique, basé sur la résonance plasmonique de surface (SPR), avec des essais de phosphorylation in vitro permit la sélection de cibles AMPK isoforme spécifique. Une de ces cibles fut la fumarate hydratase, dont la phosphorylation préférentielle par l'AMPK221 provoque une augmentation de l'efficacité enzymatique in vitro. Finalement, une classe de candidats d'interaction, les glutathion S-transferases GSTM1 et -P1, fut caractérisée en détail par un panel de méthodes d'interactomique (SPR, double hybride, co-immunoprécipitation). Cette étude les identifie comme interacteurs fiables à haute affinité ainsi que nouveaux substrats de l'AMPK. Dans le cas de GSTP1 la phosphorylation par AMPK provoque une augmentation de son activité enzymatique suggérant un rôle direct de la signalisation par AMPK dans la défense contre le stress oxydatif.

Infrared light (IR) was first discovered by Friedrich Wilhelm Herschel in 1800. However, until 1940’s, molecular IR studies involved only water and small organic molecules, because of the long measurement times. Development Fourier transform infrared spectroscopy (FTIR) has minimized the time required to obtain data, making it possible to investigate bigger biological systems, e.g. proteins and nucleic acids.This thesis concentrates on the applications of different IR spectroscopic techniques to a variety of biological systems and development of new approaches to study complicated biological events. The first paper in this work concerns using so-called caged compounds to study the aggregation of Alzheimer’s Aβ-peptide which is linked to the formation of neurotoxic fibrils in the brain. By adding caged-sulfate to the Aβ samples we were able to change the pH of the sample, while recording IR data and study fibril formation in a time-resolved manner. Then we used caged–ADP to study the production of ATP and creatine, mediated by creatine kinase (CK). Using CK as a helper enzyme we studied the effects of the phosphate binding on the secondary structure of SR Ca2+ATPse and determined the structural differences between two similar states Ca2E1ADP and Ca2E1ATP. In the second part of the thesis we used ATR-FTIR spectroscopy and a specially designed dialysis setup, to develop a general method to detect ligand binding events by observing the IR absorbance changes in the water hydration shell around the molecules. The same method was used to determine the binding of DNA to the transcription factors of the E2F family. E2F proteins play main part in the gene regulatory networks that control cell development. However how they recognize their DNA-binding sites and the mechanism of binding is not well understood. By using ATR-FTIR, we observed the changes in the secondary structure of the proteins, as well as the distortions to the DNA upon E2F-DNA complex formation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

The purpose of this study was to determine the effects of high intensity eccentric muscle damage on plasma creatine kinase and plasma malondialdehyde. Twelve subjects, who served as their own control, performed 10 sets of eccentric knee extensions at a 10 RM intensity with their dominant leg. The subjects lowered the resistance in slow controlled manner to a three count of a metronome set at one beat per second. The resistance was set at an intensity equal to 120% of the subject's concentric 1RM. Creatine kinase and malondialdehyde were measured pre-exercise and at 24, 48, 72, 96 and 120 hours post-exercise. Results (p<_ .05) showed a significant increase in both creatine kinase and malondialdehyde compared to baseline. Creatine kinase showed significant increases through 120 hours post-exercise, and peaked at 96 hours post-exercise. Malondialdehyde showed significant increases through 72 post-exercise and peaked at 24 hours postexercise. It appears that a single bout, of high intensity, eccentric exercise can cause significant increases in creatine kinase and malondialdehyde. / Department of Speech Pathology & Audiology

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 55-58). / ix, 58 leaves, bound ill. 29 cm

There is a large body of clinical and experimental evidence linking heart disease with impairment of myocardial energetics, particularly the creatine kinase (CK) system. The goal of the experiments described in this thesis was to develop and study models of increased CK phosphotransfer, by overexpressing the CK isoenzymes and/or augmenting intracellular creatine stores. Pilot experiments were performed in cultured cells, which were used to (a) study the effects of CK overexpression in vitro, and (b) validate constructs prior to generation of transgenic mice. Expression was verified at the protein level for all constructs in HL-1 and HEK293 cells, and enzymatic activity was confirmed. Mitochondrial CK (CKmt) was expressed in the mitochondria, as expected, and CKmt overexpression was associated with a significant reduction in cell death in a model of ischemia/reperfusion injury (68.1 &plusmn; 7.1% of control, p&le;0.05). Transgenic mice overexpressing CKmt in the heart were generated by a targeted approach, using PhiC31 integration at the ROSA26 locus. Transgene expression was confirmed in vitro in embryonic stem cells, and in vivo at the mRNA and protein levels. There was only a modest increase in CKmt activity; therefore, homozygous transgenic mice were generated to increase expression levels, and had 27% higher CKmt activity than wild-types (p&le;0.01). Mitochondrial localization of CKmt was confirmed by electron microscopy. Citrate synthase activity, a marker of mitochondrial volume, was ~10% lower in transgenic mice (p&le;0.05). Baseline phenotyping studies found that CKmt-overexpressing mice have normal cardiac structure and function. These mice are currently being backcrossed onto a pure C57BL/6 background for further studies in models of heart disease. In addition to CKmt, transgenic mice overexpressing the cytosolic CK isoenzymes, CK-M and CK-B, were generated. Due to the modest level of expression observed at ROSA26, random-integration transgenesis was used, and multiple lines were generated for each construct (carrying 2 or 6 transgene copies in the CK-M line; 2, 3, or ~30 in CK-B). Transgene expression was validated at the mRNA, protein, and activity levels. These lines are currently being expanded for further validation and phenotyping studies. Previous experiments in our group have demonstrated that increasing intracellular creatine (Cr) reduces ischemia/reperfusion injury, and a series of in vitro experiments was performed to determine whether this effect may be mediated by inhibition of the mitochondrial permeability transition pore (mPTP). The mPTP plays a significant role in ischemia/reperfusion, and there is evidence linking the CK system to regulation of the mPTP. Therefore, a model was developed to test whether Cr affects mPTP opening in cardiac-derived HL-1 cells, as this mechanism may contribute to the protective effect observed in vivo. Cr incubation conditions were determined empirically, and 24-hour incubation with 5mM or 10mM Cr was found to significantly delay mPTP opening, to a similar degree to the established mPTP inhibitor, cyclosporin A. This provides evidence that Cr may exert protective effects in the heart by a variety of mechanisms, in addition to its traditional role in energy metabolism. In summary, the experiments conducted in this thesis have produced a range of tools for studying augmentation of the creatine kinase system as a therapeutic target in heart disease. The results of in vitro assays indicate that mitochondrial CK may be a particularly promising target, and that inhibition of the mitochondrial permeability transition pore may contribute to the cardioprotective effect of creatine. Finally, the transgenic models generated and validated over the course of this project will allow for a wide range of future studies into the potential benefits of CK overexpression in the mammalian heart.