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).

The purpose of this thesis was to determine the combined effects of protein, creatine, and conjugated linoleic acid (CLA) supplementation during resistance training. These nutritional supplements are popular during resistance training and we wanted to determine if they would have additive effects for improving body composition and strength. Forty-four participants (32 males, 12 females, mean age 20y) were randomized into three groups to receive: 1) 36 g/d protein (PRO), 2) protein and 9 g/d creatine (PRO/CR), or 3) protein, creatine and 6 g/d CLA (PRO/CR/CLA) for 5 weeks while resistance training on a four-day cycle (three days of resistance training, followed by one day of rest). Measurements at pre- and post-testing included body composition, muscle thickness of the elbow and knee flexors and extensors, and bench and leg press strength. There were time main effects (p<0.01) for strength, and muscle thickness. The PRO/CR/CLA group had significant increases in knee extensor muscle thickness over time compared to the other groups (p<0.05). There were no other differences between groups over time. The combinations of creatine and protein, or creatine, protein and CLA had no effects on body composition. It is concluded that combining protein, creatine, and CLA has minimal effects on muscular strength, muscle thickness, and body composition.

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.

Biological tissues were studied with synchrotron infrared (IR) microspectroscopy, a technique that allows the spatially resolved determination and mapping of multiple components in situ at high spatial resolution. The first project involved studying brain tissue from TgCRND8 mice, a transgenic model of Alzheimer’s disease (AD). AD is the main cause of dementia in the ageing population, marked by the deposition of plaques composed of the Aβ peptide. Dense-cored and diffuse plaques were IR mapped and the results correlated with histochemistry and immunostaining. Spectral analysis confirmed that congophilic plaque cores were composed of highly aggregated protein in a β-sheet conformation. The amide I maximum of plaque cores was 1623 cm-1; there was no evidence of the high frequency (1680-1690 cm-1) peak seen in in vitro Aβ fibrils and attributed to anti-parallel β-sheet. A significant elevation in phospholipids was found around dense-cored plaques in TgCRND8 mice ranging in age from 5 to 21 months. This was due to an increase in cellular membranes from dystrophic neurites and glial cells around the core, but could also contribute to Aβ aggregation through the interaction of newly secreted Aβ with phospholipids. In contrast, diffuse plaques were not associated with infrared detectable changes in protein secondary structure or relative concentrations of other tissue components. In addition, focally elevated deposits of creatine, a molecule with a crucial role in energy metabolism, were discovered in AD brain tissue with IR microspectroscopy. The creatine deposits may be a previously undiscovered disease marker. A second project was part of a larger Natural Sciences and Engineering Research Council Collaborative Health Research Project (NSERC-CHRP) to test the hypothesis that treatment with anti-oxidants, L-2-oxo-thiazolidine-4-carboxylate (OTC) and quercetin, following spinal surgery may reduce oxidative stress, inflammation, and scarring. The effect of OTC and quercetin on scar tissue formation was evaluated in rats that had undergone laminectomy. Synchrotron IR microspectroscopy data were collected on scar tissue from OTC, quercetin and saline (control) treated animals, sacrificed at 3 and 21 days post-surgery. Spectral differences could be correlated with the stages of wound healing. / May 2007

The purpose of this thesis was to determine the combined effects of protein, creatine, and conjugated linoleic acid (CLA) supplementation during resistance training. These nutritional supplements are popular during resistance training and we wanted to determine if they would have additive effects for improving body composition and strength. Forty-four participants (32 males, 12 females, mean age 20y) were randomized into three groups to receive: 1) 36 g/d protein (PRO), 2) protein and 9 g/d creatine (PRO/CR), or 3) protein, creatine and 6 g/d CLA (PRO/CR/CLA) for 5 weeks while resistance training on a four-day cycle (three days of resistance training, followed by one day of rest). Measurements at pre- and post-testing included body composition, muscle thickness of the elbow and knee flexors and extensors, and bench and leg press strength. There were time main effects (p<0.01) for strength, and muscle thickness. The PRO/CR/CLA group had significant increases in knee extensor muscle thickness over time compared to the other groups (p<0.05). There were no other differences between groups over time. The combinations of creatine and protein, or creatine, protein and CLA had no effects on body composition. It is concluded that combining protein, creatine, and CLA has minimal effects on muscular strength, muscle thickness, and body composition.

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

Biological tissues were studied with synchrotron infrared (IR) microspectroscopy, a technique that allows the spatially resolved determination and mapping of multiple components in situ at high spatial resolution. The first project involved studying brain tissue from TgCRND8 mice, a transgenic model of Alzheimer’s disease (AD). AD is the main cause of dementia in the ageing population, marked by the deposition of plaques composed of the Aβ peptide. Dense-cored and diffuse plaques were IR mapped and the results correlated with histochemistry and immunostaining. Spectral analysis confirmed that congophilic plaque cores were composed of highly aggregated protein in a β-sheet conformation. The amide I maximum of plaque cores was 1623 cm-1; there was no evidence of the high frequency (1680-1690 cm-1) peak seen in in vitro Aβ fibrils and attributed to anti-parallel β-sheet. A significant elevation in phospholipids was found around dense-cored plaques in TgCRND8 mice ranging in age from 5 to 21 months. This was due to an increase in cellular membranes from dystrophic neurites and glial cells around the core, but could also contribute to Aβ aggregation through the interaction of newly secreted Aβ with phospholipids. In contrast, diffuse plaques were not associated with infrared detectable changes in protein secondary structure or relative concentrations of other tissue components. In addition, focally elevated deposits of creatine, a molecule with a crucial role in energy metabolism, were discovered in AD brain tissue with IR microspectroscopy. The creatine deposits may be a previously undiscovered disease marker. A second project was part of a larger Natural Sciences and Engineering Research Council Collaborative Health Research Project (NSERC-CHRP) to test the hypothesis that treatment with anti-oxidants, L-2-oxo-thiazolidine-4-carboxylate (OTC) and quercetin, following spinal surgery may reduce oxidative stress, inflammation, and scarring. The effect of OTC and quercetin on scar tissue formation was evaluated in rats that had undergone laminectomy. Synchrotron IR microspectroscopy data were collected on scar tissue from OTC, quercetin and saline (control) treated animals, sacrificed at 3 and 21 days post-surgery. Spectral differences could be correlated with the stages of wound healing.

Biological tissues were studied with synchrotron infrared (IR) microspectroscopy, a technique that allows the spatially resolved determination and mapping of multiple components in situ at high spatial resolution. The first project involved studying brain tissue from TgCRND8 mice, a transgenic model of Alzheimer’s disease (AD). AD is the main cause of dementia in the ageing population, marked by the deposition of plaques composed of the Aβ peptide. Dense-cored and diffuse plaques were IR mapped and the results correlated with histochemistry and immunostaining. Spectral analysis confirmed that congophilic plaque cores were composed of highly aggregated protein in a β-sheet conformation. The amide I maximum of plaque cores was 1623 cm-1; there was no evidence of the high frequency (1680-1690 cm-1) peak seen in in vitro Aβ fibrils and attributed to anti-parallel β-sheet. A significant elevation in phospholipids was found around dense-cored plaques in TgCRND8 mice ranging in age from 5 to 21 months. This was due to an increase in cellular membranes from dystrophic neurites and glial cells around the core, but could also contribute to Aβ aggregation through the interaction of newly secreted Aβ with phospholipids. In contrast, diffuse plaques were not associated with infrared detectable changes in protein secondary structure or relative concentrations of other tissue components. In addition, focally elevated deposits of creatine, a molecule with a crucial role in energy metabolism, were discovered in AD brain tissue with IR microspectroscopy. The creatine deposits may be a previously undiscovered disease marker. A second project was part of a larger Natural Sciences and Engineering Research Council Collaborative Health Research Project (NSERC-CHRP) to test the hypothesis that treatment with anti-oxidants, L-2-oxo-thiazolidine-4-carboxylate (OTC) and quercetin, following spinal surgery may reduce oxidative stress, inflammation, and scarring. The effect of OTC and quercetin on scar tissue formation was evaluated in rats that had undergone laminectomy. Synchrotron IR microspectroscopy data were collected on scar tissue from OTC, quercetin and saline (control) treated animals, sacrificed at 3 and 21 days post-surgery. Spectral differences could be correlated with the stages of wound healing.

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