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Les pathologies cardiovasculaires sont responsables d’un grand nombre de décès dans le monde aujourd’hui, dont plus de la moitié trouve son origine dans des anomalies rythmiques. Les phénomènes de dépolarisation et de repolarisation rythmiques des cardiomyocytes, à l’origine de l’activité contractile rythmique du myocarde, mettent en jeu des processus cellulaires consommateurs d’énergie et les mitochondries,. Dans cette thèse, la régulation de la fonction mitochondriale a été étudiée lors de l’induction d’arythmies ventriculaires sur coeurs isolés perfusés de rats. Les résultats montrent que l'évolution de la consommation d’oxygène du myocarde est un paramètre important dans la réponse aux arythmies. L’inhibition des myosines ATPases par la blebbistatine induit sur la durée une diminution de la susceptibilité vis-à-vis des arythmies. Les acteurs impliqués dans les différents types de réponses mitochondriales ont été caractérisés sur fibres perméabilisées. Nous montrons que la régulation de la fonction mitochondriale varie en fonction de la chambre cardiaque considérée. Les mesures de Km de la respiration mitochondriale, ainsi que l’étude des principaux mécanismes de transfert énergétiques, mettent en évidence des différences marquées entre les différentes chambres cardiaques. .Les résultats de nos travaux suggèrent que le remodelage myocardique observé en réponse aux modifications de charge, et/ou lors du développement des pathologies du myocarde sont susceptibles de favoriser la constitution d’un substrat arythmogène, propre à chaque chambre, et impliquant des perturbations des mécanismes de transfert énergétique. / Cardiovascular disease is the first cause of mortality in the whole world. Half of this mortality is due to heart failure, a progressive deterioration of cardiac contraction, which can be caused by electrical dyssynchrony. Implication of heart energetics on susceptibility to arrhythmia is therefore of particular interest to better understood the overall effect as well as the relative importance of the potential partners on developed pathologies. In this thesis, energetic regulation of mitochondria was studied in perfused hearts paced to induce arrhythmia. Myocardial oxygen consumption was shown as a crucial parameter in the response to rhythm troubles. Inhibitions of ATPase myofibrils by blebbistatin or AK-phosphotransfer by Ap5a did not produce same responses of mitochondrial compartment. When inhibited by Blebb, most of hearts presented less ectopic beats highlighting an anti-arrhythmic profile in this case of myofibrils inhibition. Energetic parameters involved in mitochondrial responses were characterized in skinned fibres. Mitochondria regulation was different between ventricle and atria, with a less sensitivity of mitochondria in atria compared to ventricle. Mechanisms of energy transfer between supply and demand were different between ventricle and atria. The role of AK-phosphotransfer was described as a determining parameter for atria energy maintenance, whereas ventricle presented a functional efficacy of CK pathway to regulate adenine nucleotide turnover. As suggested by these results, the role of adenylic nucleotides channeling could be considered as a mechanism used to counteract altered cardiac energetics that potentially sustained electrical pathologies.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / "May 2005" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Made available in DSpace on 2018-06-27T18:56:01Z (GMT). No. of bitstreams: 2 Amanda Lima de Oliveira.pdf: 513074 bytes, checksum: 393de3a3c8893cb2dad143281cc76ca4 (MD5) Amanda Lima de Oliveira.pdf.jpg: 3435 bytes, checksum: d98e4a9d95435991a70b3e949f124696 (MD5) Previous issue date: 2012-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cyclodextrins (CDs) are cyclic oligosaccharides formed by 6 (αCD), 7 (bCD) or 8 (γCD) glucose units with an internal hydrophobic cavity and outside surface hydrophilic. These three derivatives, the b-cyclodextrin (bCD) seems to be the most advantageous for pharmaceutical use for their availability, cavity size and low cost. The CDs have a future quite promising for their properties as greater absorption of drugs through the biological barriers and time of release, however, some types may not be considered non-toxic. The objective of this study was to investigate the intraperitoneal administration of βCD, M-β-CD and HP-ß-CD for 8 weeks with administered dose of 65.65 mg of CDs/kg rats/day, on parameters of biochemical analyzes, enzymes of energy metabolism, enzymes tiolicas sensitive to increase reactive oxygen species and to make this relationship, also evaluate parameters of oxidative stress in cerebral cortex, liver, kidneys and heart of wistar rats. The results showed that for the group treated with βCD there has been a significant increase in serum urea and creatinine levels, indicating nephrotoxicity, however not related to the other parameters. There was also a great reduction in serum levels of iron for the 3 CDs. The heart showed a reduction in the activity of CKmitocondrial and increase for AK by M-β-CD and reduction of CKmit by HP-ß-CD, but showed a reduction in the levels of diclorofluorceina (DCF) to the 3 CDs and protein carbonyl) by βCD. For the rim there was no significant change in comreducao activity of CKmit by HP-β-CD. In liver tissue, the βCD and M-β-CD reduced the activity of PK, but this is not reflected in blood glucose levels. In the cerebral cortex, the βCD reduced the activity of enzymes CK mitochondrial and PK, also reduced TBARS, but increased carbonyl protein. The indices lipidemic reduced reported by other researchers was not observed in this work, because the group of M-β- CD has a significant increase in serum levels of LDL cholesterol, in addition to aspartate aminostransferase AST, albumin, total protein, alkaline phosphatase, sodium, calcium, magnesium and phosphate. Our results indicate that some CDs alter enzymes crucial for energy metabolism, mainly of brain tissue with a reduction in activity and the PK by βCD. If changes in the activity of these enzymes occur in people who use drugs by intraperitoneal route, it is possible that the energy metabolism and brain functioning may be affected causing damage to the tissue. However more studies are needed to elucidate how there was a reduction of serum iron and as the cyclodextrins affect a structure so well protected by blood-brain barrier as the brain. / As ciclodextrinas (CDs) são oligossacarídeos cíclicos formados por 6 (αCD), 7 (bCD) ou 8 (γCD) unidades de glicose com uma cavidade interna hidrofóbica e superfície externa hidrofílica. Destes três derivados, a b-ciclodextrina (bCD) parece ser a mais vantajosa para utilização farmacêutica pela sua disponibilidade, tamanho da cavidade e baixo custo. O interesse pelas CDs se dá pelas suas propriedades como maior absorção dos fármacos através das barreiras biológicas e tempo de liberação, entretanto, alguns tipos não podem ser considerados atóxicas. O objetivo deste estudo foi investigar a administração intraperitoneal de βCD (Beta Ciclodextrina), M-β-CD (Metil Beta Ciclodextrina) e HP-β-CD (Hidroxypropil Beta Ciclodextrina) durante 8 semanas com dose administrada de 65,65 mg das CDs/kg rato/dia, sobre parâmetros de análises bioquímicas, de enzimas do metabolismo energético, enzimas tiólicas sensíveis ao aumento de espécies reativas e para fazer a relação, também avaliar parâmetros de estresse oxidativo em córtex cerebral, fígado, rins e coração de ratos wistar. Os resultados mostraram que para o grupo tratado com βCD houve um aumento significativo nos níveis séricos de uréia e creatinina, indicando nefrotoxidade, porém não relacionada com os demais parâmetros. Também houve uma grande redução nos níveis séricos de ferro para as 3 CDs. O coração apresentou redução na atividade da Creatinaquinase mitocondrial (CKmit) e aumento para Adenilatoquinase (AK) pela M- β-CD e redução da CKmit pela HP-β-CD, porém apresentou uma redução nos níveis de diclorofluoresceína (DCF) para as 3 CDs e carbonilas proteicas pela βCD. Para o rim houve alteração significativa com redução na atividade da CKmit pela HP-β-CD. No tecido hepático, a βCD e M-β-CD reduziram a atividade da Piruvatoquinase (PK), porém isto não refletiu nos níveis glicêmicos. No córtex cerebral, a βCD reduziu a atividade das enzimas CK mitocondrial e PK, também reduziu TBARS (Espécies reativas ao ácido tiobarbitúrico), mas aumentou carbonilas proteicas. Os índices lipidêmicos reduzidos relatados por outros pesquisadores não foi observado neste trabalho, pois o grupo da M-β-CD apresentou um aumento significativo nos níveis séricos de LDL (lipoproteína de baixa densidade), além de AST (aspartato aminostransferase), albumina, proteínas totais, fosfatase alcalina, sódio, cálcio, magnésio e fosfato. Os resultados indicam que algumas CDs alteram enzimas cruciais do metabolismo energético, principalmente do tecido cerebral com redução na atividade da PK pela βCD. Possíveis alterações na atividade destas enzimas podem afetar o metabolismo energético e o funcionamento cerebral causando dano ao tecido. Entretanto mais estudos são necessários para elucidar de que forma ocorreu a redução sérica de ferro e como as ciclodextrinas afetaram uma estrutura tão bem protegida pela barreira hemato-encefálica como a cerebral.

The work in this thesis focuses on the study of three main aspects of proteins, viz, Protein structure, stability, and dynamics. Chapter 1 is a general introduction to the topics studied in this thesis. Chapter 2 deals with the first aspect, i.e., protein structure in which we describe an approach to use saturation mutagenesis phenotypes to guide protein structure prediction. Chapters 3 and 4 discuss how to increase protein stability using surface electrostatics, and Chapter 5 details a method to predict whether a proline substitution in a given protein would be stabilizing or destabilizing. Hence, Chapters 3-5 can be associated with the second aspect, i.e., protein stability. The third aspect, namely protein dynamics, is dealt with in Chapters 6 and 7 which study conformational dynamics of adenylate kinase. Protein structure prediction is a difficult problem with two major bottlenecks, namely, generation of accurate models and the selection of the most appropriate models from a large pool of decoys. In Chapter 2, the problem of model discrimination is addressed using mutant phenotype information derived from saturation mutagenesis library. A library of ~1500 single-site mutants of the E. coli toxin CcdB (Controller of Cell Division or Death B) has been previously constructed in our lab. The pooled library was characterized in terms of individual mutant phenotypes at various expression levels which were derived from the relative populations of mutants at each expression level. The relative populations of mutants were estimated using deep sequencing. Mutational tolerances were derived from the phenotypic data and were used to define an empirical parameter which correlated with a structural parameter, residue depth. We further studied how this new parameter can be used for model discrimination. Increasing protein stability in a rational way is a challenging problem and has been addressed by various approaches. One of the most commonly used approaches is optimization of protein core residues. Recently, optimization of protein surface electrostatics has been shown to be a useful approach for increasing stability of proteins. In Chapter 3, from analyses of a dataset of ~1750 non-homologues proteins, we show that proteins having a pI away from physiological pH, possess a significant fraction of unfavorably placed charged amino acids on their surface. One way to increase protein stability in such cases might be to alter these surface charges. This hypothesis was validated experimentally by making charge reversal mutations at putative unfavorable positions on the surface of maltose binding protein, MBP. The observed stabilization can potentially be increased by combining multiple individually stabilizing mutations. Different combinations of such mutations were made and tested in Chapter 4 to decide which mutants can be combined to achieve net stabilization. Ideas were tested through systematic experimentation which involved generation of two-site, three-site, and four-site mutations. A maximum increase in melting temperature (Tm) of 3-4 °C over wild-type protein was achieved upon combination of individually stabilizing mutants. Proline (Pro) has two special stereo-chemical properties when it is a part of a polypeptide chain. First the φ value of Pro has a very constrained distribution and second, Pro lacks an amide hydrogen. Due to these properties, introduction of Pro might perturb stability/activity of the protein. In Chapter 5 we describe a procedure to accurately predict the effects of Pro introduction on protein stability. Pro scanning mutagenesis was carried out on the model protein CcdB and the in vivo activity of the individual mutants was also examined. A decision tree was constructed, using the special stereo-chemical properties of Pro to maximize correlation of predicted phenotype with the in vivo activity. Binary classification as perturbing or non-perturbing of every Pro substitution was possible using the decision tree. The performance of the decision tree was assessed on various test systems, and the average accuracy was found to be ~75%. The role of conformational dynamics in enzyme catalysis has been explored in great detail in the literature. In Chapter 6, with the help of very long (350 ns), fully atomistic, explicit solvent molecular dynamics simulations, we studied conformational dynamics of adenylate kinase. We found the existence of a relatively stable state which lies intermediate between the open and closed conformations of the enzyme. The finding was further confirmed by computing a two dimensional configurational free energy surface when motions along each of the two movable domains (LID and NMP) are considered as reaction coordinates. We also discussed possible roles of the intermediate state during enzyme catalysis. The role of water in stabilization of intermediate states was also discussed. In Chapter 7, we studied dynamical coupling between LID and NMP domains of adenylate kinase during domain opening. Our observation suggests that the LID domain should start opening prior to the NMP domain. On the domain opening trajectory, the free energy surface of LID domain was found to be very rugged. We discuss a possible role of water in the ruggedness of the domain motions. The Appendix contains 3 supplementary parts of the thesis. Appendix I is a mutant dataset obtained from 454 sequencing analysis. It includes the normalized number of reads per mutation at each expression level along with mutational sensitivity score. Appendix II is parameters used for one of the electrostatic calculations. Appendix III contains a list of PDB ids used for database analysis in surface electrostatics work discussed in Chapter 3.

Right ventricular function (RV) is one of the most important predictors of prognosis in many cardiovascular disease states. Despite the significance of RV function to survival, there are no therapies that directly nor selectively improve RV function. As well, the basis for RV failure is poorly understood. This is particularly relevant for patients with pulmonary arterial hypertension (PAH), where RV failure in the setting of pressure overload is the leading cause of death. PAH will be introduced in the 2nd chapter of this thesis by comparing and refining contemporary mortality risk assessment strategies. I will then explore 1) RV neurohormonal function and, 2) RV energetics, two molecular pathways thought to be involved in the pathogenesis and progression of maladaptive RV failure. I employed small animal molecular imaging using positron emission tomography (PET) to non-invasively investigate these pathways. The PET imaging techniques employed in this thesis have the unique potential for translation to human studies, to further explore disease mechanisms.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21265号 / 医科博第92号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 松田 道行, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The fitness function developed in this thesis directly links the physicochemical properties of an enzyme to evolutionary fates in a quantitative and predictive manner through a comparative study of empirical and simulated data. The success or failure of organisms during evolution is dictated by changes in molecular structure that give rise to changes in fitness revealed by evolutionary dynamics within a population. While the conceptual link between genotype, phenotype and fitness is clear, the ability to relate these in a quantitative manner remains difficult. I show here that predicting success during adaptation can depend critically upon enzyme kinetic and folding models. We used a 'weak link' method to favor mutations to an essential, but maladapted adenylate kinase gene within a microbial population that resulted in the identification of five mutants that arose nearly simultaneously and competed for success. The unique catalytic role of adenylate kinase in vivo is to maintain adenylate homeostasis by catalyzing the reaction: ATP + AMP [imaginary] ADP. The stabilizing substitutions retained this essential function and were shown to be necessary for viability at higher temperatures. Physicochemical characterization of these mutants demonstrated that, although steady-state enzyme activity is important, success within the population is critically dependent on resistance to denaturation and aggregation thus emphasizing the importance of proper folding in adaptation. In vitro activity is a product of critical catalytic and folding pathways, and hence is a valuable proxy for fitness. A fitness function relating in vitro measurements of enzyme activity and reversible and irreversible unfolding to growth rate must impose an activity threshold above which there is no added fitness benefit in order to reproduce in vivo evolutionary fates in an in silico population. The fitness function thereby links organismal adaptation to the properties of a single gene. Understanding the physical basis for adaptation of an organism is the first step in the development of approaches that can accurately model, and someday predict, the manner in which organisms would respond to new antibiotics and improve upon the current clinical regimens.

The first type of dynein identified, axonemel dynein (Gibbons and Rowe, 1965), slides adjacent microtubules within the axoneme, generating the force necessary for ciliary and flagellar beating. The outer dynein arm is an important component of the flagellar axoneme, providing up to 60% of the force for flagellar motility. In the absence of the outer arm, cells swim with a slow-jerky motion at about 1/3rd the speed of wild-type cells, and the flagellar beat frequency is markedly reduced. Sixteen genes (ODA1-ODA16) have been identified that are required for outer arm assembly in Chlamydomonas reinhardtii. In addition, PF13, PF22, and FLA14 are required for outer dynein arm assembly, but their phenotypes are pleiotropic, suggesting that they affect additional flagellar components. Of the uncloned genes, ODA5, ODA8, and ODA10 are of particular interest because they do not encode subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC). Mutant alleles of these genes are unable to complement in temporary dikaryons, suggesting that the gene products interact with each other (Kamiya, 1988). Since the genes encoding all of the known components of the outer dynein arm and the ODA-DC have been characterized, it is of great interest to identify the gene products of these additional, uncloned ODA alleles. The first chapter provides an introduction to the Chlamydomonasflagellum, the dyneins in general, the outer dynein arm in particular, and mutations that impinge on the assembly and regulation of this important axonemal structure. The second chapter addresses the identification and isolation of genomic DNA containing the ODA5 gene. Utilizing a NIT1-tagged oda5-insertional mutant, I identified sequences flanking the site of the inserted NIT1 gene. These sequences were used to isolate wild-type genomic clones spanning the ODA5 gene. When transformed into the oda5 mutant, the wild-type clones rescued the mutant phenotype. These results demonstrated the successful isolation of the ODA5 gene. The third chapter describes the identification of the ODA5 gene and its corresponding cDNA. The rescuing genomic fragments were sequenced. Gene modeling was used to predict intron-exon splice sites. Primers to predicted exons were designed and used to obtain the ODA5 cDNA. The gene structure of Oda5 was analyzed and its predicted amino acid sequence deduced. Secondary structure predictions indicate that Oda5p is likely to contain a series of coiled-coil domains, followed by a poly-glycine sequence and a short, highly charged region. Northern analysis demonstrated that ODA5 gene expression is upregulated by deflagellation, a hallmark of many flagellar mRNAs. Data in CHAPTER IV further characterize the Oda5 protein and its association with the axoneme. Oda5p localizes to the flagellum, consistent with the enhancement in mRNA levels in response to deflagellation. Within the flagellum, Oda5p is an axonemal component that is released from the axoneme upon high salt extraction, as are the ODA-DC and the outer dynein arm. However, Oda5p does not associate with this super-complex in the high salt extract as determined by sucrose gradient sedimentation. Oda5p assembles onto the axoneme independently of the outer dynein arm and the ODA-DC,demonstrating it does not require these complexes for localization. Furthermore, Oda5p assembles onto the axoneme in the oda8, but not the oda10 mutant, demonstrating a role for the Oda10 protein in localization of Oda5p. These data provide the first biochemical evidence for an interaction between Oda5p and Oda10p. CHAPTER V reveals the discovery of a previously unrecognized phenotype exhibited in both oda5 and oda10 mutant strains: a defect in the assembly of a previously unknown flagellar adenylate kinase (AK). The protein levels of this flagellar AK are reduced in oda5 mutant axonemes, as determined by quantitative mass spectrometry. Direct enzymatic assays confirmed a reduction in AK activity in both oda5 and oda10 mutant axonemes, providing a second line of biochemical evidence supporting a complex containing Oda5p and OdalOp. The sequence of the flagellar AK gene and its cDNA were determined. CHAPTER VI details our efforts to identify the ODA10 gene. Genomic clones were isolated, which contain sequences at, or near, the ODA10 locus. Analysis of the genomic clones yielded no insights into the identity of the ODA10 gene. The inability of these clones to rescue the Oda10-motility phenotype indicates that these clones most likely do not contain an intact ODA10 gene. And lastly, CHAPTER VII discusses future experimentation that can be done based on the data provided by the current study.

abstract: In a typical living cell, millions to billions of proteins—nanomachines that fluctuate and cycle among many conformational states—convert available free energy into mechanochemical work. A fundamental goal of biophysics is to ascertain how 3D protein structures encode specific functions, such as catalyzing chemical reactions or transporting nutrients into a cell. Protein dynamics span femtosecond timescales (i.e., covalent bond oscillations) to large conformational transition timescales in, and beyond, the millisecond regime (e.g., glucose transport across a phospholipid bilayer). Actual transition events are fast but rare, occurring orders of magnitude faster than typical metastable equilibrium waiting times. Equilibrium molecular dynamics (EqMD) can capture atomistic detail and solute-solvent interactions, but even microseconds of sampling attainable nowadays still falls orders of magnitude short of transition timescales, especially for large systems, rendering observations of such "rare events" difficult or effectively impossible. Advanced path-sampling methods exploit reduced physical models or biasing to produce plausible transitions while balancing accuracy and efficiency, but quantifying their accuracy relative to other numerical and experimental data has been challenging. Indeed, new horizons in elucidating protein function necessitate that present methodologies be revised to more seamlessly and quantitatively integrate a spectrum of methods, both numerical and experimental. In this dissertation, experimental and computational methods are put into perspective using the enzyme adenylate kinase (AdK) as an illustrative example. We introduce Path Similarity Analysis (PSA)—an integrative computational framework developed to quantify transition path similarity. PSA not only reliably distinguished AdK transitions by the originating method, but also traced pathway differences between two methods back to charge-charge interactions (neglected by the stereochemical model, but not the all-atom force field) in several conserved salt bridges. Cryo-electron microscopy maps of the transporter Bor1p are directly incorporated into EqMD simulations using MD flexible fitting to produce viable structural models and infer a plausible transport mechanism. Conforming to the theme of integration, a short compendium of an exploratory project—developing a hybrid atomistic-continuum method—is presented, including initial results and a novel fluctuating hydrodynamics model and corresponding numerical code. / Dissertation/Thesis / Doctoral Dissertation Physics 2017