Conceptual framework for strengthening exercises to prevent hamstring strains

High-speed running accounts for the majority of hamstring strains in many sports. The terminal swing phase is believed to be the most hazardous as the hamstrings are undergoing an active lengthening contraction in a long muscle length position. Prevention-based strength training mainly focuses on eccentric exercises. However, it appears crucial to integrate other parameters than the contraction type. Therefore, the aim of this study is to present a conceptual framework based on six key parameters (contraction type, load, range of motion, angular velocity, uni-/bilateral exercises, kinetic chain) for the hamstring's strength exercise for strain prevention. Based on the biomechanical parameters of sprinting, it is proposed to use high-load eccentric contractions. The movement should be performed at a slow to moderate angular velocity and focused at the knee joint, while the hip is kept in a large flexion position in order to reach a greater elongation stress of the hamstrings than in the terminal swing phase. In this way, we believe that, during sprinting, athletes would be better trained to brake the knee extension effectively in the whole range of motion without overstretch of the hamstrings. Finally, based on its functional application, unilateral open kinetic chain should be preferred.

A regulatory network for the efficient control of transgene expression.

BACKGROUND: Expression of heterologous genes in mammalian cells or organisms for therapeutic or experimental purposes often requires tight control of transgene expression. Specifically, the following criteria should be met: no background gene activity in the off-state, high gene expression in the on-state, regulated expression over an extended period, and multiple switching between on- and off-states. METHODS: Here, we describe a genetic switch system for controlled transgene transcription using chimeric repressor and activator proteins functioning in a novel regulatory network. In the off-state, the target transgene is actively silenced by a chimeric protein consisting of multimerized eukaryotic transcriptional repression domains fused to the DNA-binding tetracycline repressor. In the on-state, the inducer drug doxycycline affects both the derepression of the target gene promoter and activation by the GAL4-VP16 transactivator, which in turn is under the control of an autoregulatory feedback loop. RESULTS: The hallmark of this new system is the efficient transgene silencing in the off-state, as demonstrated by the tightly controlled expression of the highly cytotoxic diphtheria toxin A gene. Addition of the inducer drug allows robust activation of transgene expression. In stably transfected cells, this control is still observed after months of repeated cycling between the repressed and activated states of the target genes. CONCLUSIONS: This system permits tight long-term regulation when stably introduced into cell lines. The underlying principles of this network system should have general applications in biotechnology and gene therapy.

Identification of Novel Craniofacial Regulatory Domains Located far Upstream of SOX9 and Disrupted in Pierre Robin Sequence.

Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ∼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ∼1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS.

Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHAO.

In Pseudomonasfluorescens strain CHAO, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere. Two Tn5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P. syringae pv. syringae as well as that of P. fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0. This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease. Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA. Independent point and deletion mutations arose at different sites in the gacA gene. In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased. In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions.

Large scale assessment of regulatory evolution and transcriptome complexity in mammals

AbstractIn addition to genetic changes affecting the function of gene products, changes in gene expression have been suggested to underlie many or even most of the phenotypic differences among mammals. However, detailed gene expression comparisons were, until recently, restricted to closely related species, owing to technological limitations. Thus, we took advantage of the latest technologies (RNA-Seq) to generate extensive qualitative and quantitative transcriptome data for a unique collection of somatic and germline tissues from representatives of all major mammalian lineages (placental mammals, marsupials and monotremes) and birds, the evolutionary outgroup.In the first major project of my thesis, we performed global comparative analyses of gene expression levels based on these data. Our analyses provided fundamental insights into the dynamics of transcriptome change during mammalian evolution (e.g., the rate of expression change across species, tissues and chromosomes) and allowed the exploration of the functional relevance and phenotypic implications of transcription changes at a genome-wide scale (e.g., we identified numerous potentially selectively driven expression switches).In a second project of my thesis, which was also based on the unique transcriptome data generated in the context of the first project we focused on the evolution of alternative splicing in mammals. Alternative splicing contributes to transcriptome complexity by generating several transcript isoforms from a single gene, which can, thus, perform various functions. To complete the global comparative analysis of gene expression changes, we explored patterns of alternative splicing evolution. This work uncovered several general and unexpected patterns of alternative splicing evolution (e.g., we found that alternative splicing evolves extremely rapidly) as well as a large number of conserved alternative isoforms that may be crucial for the functioning of mammalian organs.Finally, the third and final project of my PhD consisted in analyzing in detail the unique functional and evolutionary properties of the testis by exploring the extent of its transcriptome complexity. This organ was previously shown to evolve rapidly both at the phenotypic and molecular level, apparently because of the specific pressures that act on this organ and are associated with its reproductive function. Moreover, my analyses of the amniote tissue transcriptome data described above, revealed strikingly widespread transcriptional activity of both functional and nonfunctional genomic elements in the testis compared to the other organs. To elucidate the cellular source and mechanisms underlying this promiscuous transcription in the testis, we generated deep coverage RNA-Seq data for all major testis cell types as well as epigenetic data (DNA and histone methylation) using the mouse as model system. The integration of these complete dataset revealed that meiotic and especially post-meiotic germ cells are the major contributors to the widespread functional and nonfunctional transcriptome complexity of the testis, and that this "promiscuous" spermatogenic transcription is resulting, at least partially, from an overall transcriptionally permissive chromatin state. We hypothesize that this particular open state of the chromatin results from the extensive chromatin remodeling that occurs during spermatogenesis which ultimately leads to the replacement of histones by protamines in the mature spermatozoa. Our results have important functional and evolutionary implications (e.g., regarding new gene birth and testicular gene expression evolution).Generally, these three large-scale projects of my thesis provide complete and massive datasets that constitute valuables resources for further functional and evolutionary analyses of mammalian genomes.

CHARACTERIZATION OF NrsZ, A NOVEL SMALL REGULATORY NON-CODING RNA INVOLVED IN THE ADAPTATION OF PSEUDOMONAS AERUGINOSA PAO1

The opportunistic ubiquitous pathogen Pseudomonas aeruginosa strain PAOl is a versatile Gram-negative bacterium that has the extraordinary capacity to colonize a wide diversity of ecological niches and to cause severe and persistent infections in humans. To ensure an optimal coordination of the genes involved in nutrient utilization, this bacterium uses the NtrB/C and/or the CbrA/B two-component systems, to sense nutrients availability and to regulate in consequence the expression of genes involved in their uptake and catabolism. NtrB/C is specialized in nitrogen utilization, while the CbrA/B system is involved in both carbon and nitrogen utilization and both systems activate their target genes expression in concert with the alternative sigma factor RpoN. Moreover, the NtrB/C and CbrA/B two- component systems regulate the secondary metabolism of the bacterium, such as the production of virulence factors. In addition to the fine-tuning transcriptional regulation, P. aeruginosa can rapidly modulate its metabolism using small non-coding regulatory RNAs (sRNAs), which regulate gene expression at the post-transcriptional level by diverse and sophisticated mechanisms and contribute to the fast physiological adaptability of this bacterium. In our search for novel RpoN-dependent sRNAs modulating the nutritional adaptation of P. aeruginosa PAOl, we discovered NrsZ (Nitrogen regulated sRNA), a novel RpoN-dependent sRNA that is induced under nitrogen starvation by the NtrB/C two-component system. NrsZ has a unique architecture, formed of three similar stem-loop structures (SL I, II and II) separated by variant spacer sequences. Moreover, this sRNA is processed in short individual stem-loop molecules, by internal cleavage involving the endoribonuclease RNAse E. Concerning NrsZ functions in P. aeruginosa PAOl, this sRNA was shown to trigger the swarming motility and the rhamnolipid biosurfactants production. This regulation is due to the NrsZ-mediated activation of rhlA expression, a gene encoding for an enzyme essential for swarming motility and rhamnolipids production. Interestingly, the SL I structure of NrsZ ensures its regulatory function on rhlA expression, suggesting that the similar SLs are the functional units of this modular sRNA. However, the regulatory mechanism of action of NrsZ on rhlA expression activation remains unclear and is currently being investigated. Additionally, the NrsZ regulatory network was investigated by a transcriptome analysis, suggesting that numerous genes involved in both primary and secondary metabolism are regulated by this sRNA. To emphasize the importance of NrsZ, we investigated its conservation in other Pseudomonas species and demonstrated that NrsZ is conserved and expressed under nitrogen limitation in Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48 and Pseudomonas syringae pv. tomato DC3000, strains having different ecological features, suggesting an important role of NrsZ in the adaptation of Pseudomonads to nitrogen starvation. Interestingly the architecture of the different NrsZ homologs is similarly composed by SL structures and variant spacer sequences. However, the number of SL repetitions is not identical, and one to six SLs were predicted on the different NrsZ homologs. Moreover, NrsZ is processed in short molecules in all the strains, similarly to what was previously observed in P. aeruginosa PAOl, and the heterologous expression of the NrsZ homologs restored rhlA expression, swarming motility and rhamnolipids production in the P. aeruginosa NrsZ mutant. In many aspects, NrsZ is an atypical sRNA in the bacterial panorama. To our knowledge, NrsZ is the first described sRNA induced by the NtrB/C. Moreover, its unique modular architecture and its processing in similar short SL molecules suggest that NrsZ belongs to a novel family of bacterial sRNAs. -- L'agent pathogène opportuniste et ubiquitaire Pseudomonas aeruginosa souche PAOl est une bactérie Gram négative versatile ayant l'extraordinaire capacité de coloniser différentes niches écologiques et de causer des infections sévères et persistantes chez l'être humain. Afin d'assurer une coordination optimale des gènes impliqués dans l'utilisation de différents nutriments, cette bactérie se sert de systèmes à deux composants tel que NtrB/C et CbrA/B afin de détecter la disponibilité des ressources nutritives, puis de réguler en conséquence l'expression des gènes impliqués dans leur importation et leur catabolisme. Le système NtrB/C régule l'utilisation des sources d'azote alors que le système CbrA/B est impliqué à la fois dans l'utilisation des sources de carbone et d'azote. Ces deux systèmes activent l'expression de leurs gènes-cibles de concert avec le facteur sigma alternatif RpoN. En outre, NtrB/C et CbrA/B régulent aussi le métabolisme secondaire, contrôlant notamment la production d'importants facteurs de virulence. En plus de toutes ces régulations génétiques fines ayant lieu au niveau transcriptionnel, P. aeruginosa est aussi capable de moduler son métabolisme en se servant de petits ARNs régulateurs non-codants (ARNncs), qui régulent l'expression génétique à un niveau post- transcriptionnel par divers mécanismes sophistiqués et contribuent à rendre particulièrement rapide l'adaptation physiologique de cette bactérie. Au cours de nos recherches sur de nouveaux ARNncs dépendant du facteur sigma RpoN et impliqués dans l'adaptation nutritionnelle de P. aeruginosa PAOl, nous avons découvert NrsZ (Nitrogen regulated sRNA), un ARNnc induit par la cascade NtrB/C-RpoN en condition de carence en azote. NrsZ a une architecture unique, composée de trois structures en tige- boucle (TB I, II et III) hautement similaires et séparées par des « espaceurs » ayant des séquences variables. De plus, cet ARNnc est clivé en petits fragments correspondant au trois molécules en tige-boucle, par un processus de clivage interne impliquant l'endoribonucléase RNase E. Concernant les fonctions de NrsZ chez P. aeruginosa PAOl, cet ARNnc est capable d'induire la motilité de type « swarming » et la production de biosurfactants, nommés rhamnolipides. Cette régulation est due à l'activation par NrsZ de l'expression de rhlA, un gène essentiel pour la motilité de type swarming et pour la production de rhamnolipides. Étonnamment, la structure TB I est capable d'assurer à elle seule la fonction régulatrice de NrsZ sur l'expression de rhlA, suggérant que ces molécules TBs sont les unités fonctionnelles de cet ARNnc modulaire. Cependant, le mécanisme moléculaire par lequel NrsZ active l'expression de rhlA demeure à ce jour incertain et est actuellement à l'étude. En plus, le réseau de régulations médiées par NrsZ a été étudié par une analyse de transcriptome qui a indiqué que de nombreux gènes impliqués dans le métabolisme primaire ou secondaire seraient régulés par NrsZ. Pour accentuer l'importance de NrsZ, nous avons étudié sa conservation dans d'autres espèces de Pseudomonas. Ainsi, nous avons démontré que NrsZ est conservé et exprimé en situation de carence d'azote par les souches Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48, Pseudomonas syringae pv. tomato DC3000, quatre espèces ayant des caractéristiques écologiques très différentes, suggérant que NrsZ joue un rôle important dans l'adaptation du genre Pseudomonas envers la carence en azote. Chez toutes les souches étudiées, les différents homologues de NrsZ présentent une architecture similaire faite de TBs conservées et d'espaceurs. Cependant, le nombre de TBs n'est pas identique et peut varier de une à six copies selon la souche. Les différentes versions de NrsZ sont clivées en petites molécules dans ces quatre souches, comme il a été observé chez P. aeruginosa PAOl. De plus, l'expression hétérologue des différentes variantes de NrsZ est capable de restaurer l'expression de rhlA, la motilité swarming et la production de rhamnolipides dans une souche de P. aeruginosa dont nrsZ a été inactivé. Par bien des aspects, NrsZ est un ARNnc atypique dans le monde bactérien. À notre connaissance, NrsZ est le premier ARNnc décrit comme étant régulé par le système NtrB/C. De plus, son unique architecture modulaire et son clivage en petites molécules similaires suggèrent que NrsZ appartient à une nouvelle famille d'ARNncs bactériens.

KCM, a general framework for codon-based model of evolution

With the advancement of high-throughput sequencing and dramatic increase of available genetic data, statistical modeling has become an essential part in the field of molecular evolution. Statistical modeling results in many interesting discoveries in the field, from detection of highly conserved or diverse regions in a genome to phylogenetic inference of species evolutionary history Among different types of genome sequences, protein coding regions are particularly interesting due to their impact on proteins. The building blocks of proteins, i.e. amino acids, are coded by triples of nucleotides, known as codons. Accordingly, studying the evolution of codons leads to fundamental understanding of how proteins function and evolve. The current codon models can be classified into three principal groups: mechanistic codon models, empirical codon models and hybrid ones. The mechanistic models grasp particular attention due to clarity of their underlying biological assumptions and parameters. However, they suffer from simplified assumptions that are required to overcome the burden of computational complexity. The main assumptions applied to the current mechanistic codon models are (a) double and triple substitutions of nucleotides within codons are negligible, (b) there is no mutation variation among nucleotides of a single codon and (c) assuming HKY nucleotide model is sufficient to capture essence of transition- transversion rates at nucleotide level. In this thesis, I develop a framework of mechanistic codon models, named KCM-based model family framework, based on holding or relaxing the mentioned assumptions. Accordingly, eight different models are proposed from eight combinations of holding or relaxing the assumptions from the simplest one that holds all the assumptions to the most general one that relaxes all of them. The models derived from the proposed framework allow me to investigate the biological plausibility of the three simplified assumptions on real data sets as well as finding the best model that is aligned with the underlying characteristics of the data sets. -- Avec l'avancement de séquençage à haut débit et l'augmentation dramatique des données géné¬tiques disponibles, la modélisation statistique est devenue un élément essentiel dans le domaine dé l'évolution moléculaire. Les résultats de la modélisation statistique dans de nombreuses découvertes intéressantes dans le domaine de la détection, de régions hautement conservées ou diverses dans un génome de l'inférence phylogénétique des espèces histoire évolutive. Parmi les différents types de séquences du génome, les régions codantes de protéines sont particulièrement intéressants en raison de leur impact sur les protéines. Les blocs de construction des protéines, à savoir les acides aminés, sont codés par des triplets de nucléotides, appelés codons. Par conséquent, l'étude de l'évolution des codons mène à la compréhension fondamentale de la façon dont les protéines fonctionnent et évoluent. Les modèles de codons actuels peuvent être classés en trois groupes principaux : les modèles de codons mécanistes, les modèles de codons empiriques et les hybrides. Les modèles mécanistes saisir une attention particulière en raison de la clarté de leurs hypothèses et les paramètres biologiques sous-jacents. Cependant, ils souffrent d'hypothèses simplificatrices qui permettent de surmonter le fardeau de la complexité des calculs. Les principales hypothèses retenues pour les modèles actuels de codons mécanistes sont : a) substitutions doubles et triples de nucleotides dans les codons sont négligeables, b) il n'y a pas de variation de la mutation chez les nucléotides d'un codon unique, et c) en supposant modèle nucléotidique HKY est suffisant pour capturer l'essence de taux de transition transversion au niveau nucléotidique. Dans cette thèse, je poursuis deux objectifs principaux. Le premier objectif est de développer un cadre de modèles de codons mécanistes, nommé cadre KCM-based model family, sur la base de la détention ou de l'assouplissement des hypothèses mentionnées. En conséquence, huit modèles différents sont proposés à partir de huit combinaisons de la détention ou l'assouplissement des hypothèses de la plus simple qui détient toutes les hypothèses à la plus générale qui détend tous. Les modèles dérivés du cadre proposé nous permettent d'enquêter sur la plausibilité biologique des trois hypothèses simplificatrices sur des données réelles ainsi que de trouver le meilleur modèle qui est aligné avec les caractéristiques sous-jacentes des jeux de données. Nos expériences montrent que, dans aucun des jeux de données réelles, tenant les trois hypothèses mentionnées est réaliste. Cela signifie en utilisant des modèles simples qui détiennent ces hypothèses peuvent être trompeuses et les résultats de l'estimation inexacte des paramètres. Le deuxième objectif est de développer un modèle mécaniste de codon généralisée qui détend les trois hypothèses simplificatrices, tandis que d'informatique efficace, en utilisant une opération de matrice appelée produit de Kronecker. Nos expériences montrent que sur un jeux de données choisis au hasard, le modèle proposé de codon mécaniste généralisée surpasse autre modèle de codon par rapport à AICc métrique dans environ la moitié des ensembles de données. En outre, je montre à travers plusieurs expériences que le modèle général proposé est biologiquement plausible.

The many uses of regulatory impact assessment: A meta-analysis of EU and UK cases

Research on regulation has crossed paths with the literature on policy instruments, showing that regulatory policy instruments contain cognitive and normative beliefs about policy. Thus, their usage stacks the deck in favor of one type of actor or one type of regulatory solution. In this article, we challenge the assumption that there is a predetermined relationship between ideas, regulatory policy instruments, and outcomes. We argue that different combinations of conditions lead to different outcomes, depending on how actors use the instrument. Empirically, we analyze 31 EU and UK case studies of regulatory impact assessment (RIA) - a regulatory policy instrument that has been pivotal in the so-called better regulation movement. We distinguish four main usages of RIA, that is, political, instrumental, communicative, and perfunctory. We find that in our sample instrumental usage is not so rare and that the contrast between communicative and political usages is less stark than is commonly thought. In terms of policy recommendations, our analysis suggests that there may be different paths to desirable outcomes. Policymakers should therefore explore different combinations of conditions leading to the usages they deem desirable rather than arguing for a fixed menu of variables.

Accurate and efficient simulation of multiphase flow in a heterogeneous reservoir by using error estimate and control in the multiscale finite-volume framework

The multiscale finite-volume (MSFV) method is designed to reduce the computational cost of elliptic and parabolic problems with highly heterogeneous anisotropic coefficients. The reduction is achieved by splitting the original global problem into a set of local problems (with approximate local boundary conditions) coupled by a coarse global problem. It has been shown recently that the numerical errors in MSFV results can be reduced systematically with an iterative procedure that provides a conservative velocity field after any iteration step. The iterative MSFV (i-MSFV) method can be obtained with an improved (smoothed) multiscale solution to enhance the localization conditions, with a Krylov subspace method [e.g., the generalized-minimal-residual (GMRES) algorithm] preconditioned by the MSFV system, or with a combination of both. In a multiphase-flow system, a balance between accuracy and computational efficiency should be achieved by finding a minimum number of i-MSFV iterations (on pressure), which is necessary to achieve the desired accuracy in the saturation solution. In this work, we extend the i-MSFV method to sequential implicit simulation of time-dependent problems. To control the error of the coupled saturation/pressure system, we analyze the transport error caused by an approximate velocity field. We then propose an error-control strategy on the basis of the residual of the pressure equation. At the beginning of simulation, the pressure solution is iterated until a specified accuracy is achieved. To minimize the number of iterations in a multiphase-flow problem, the solution at the previous timestep is used to improve the localization assumption at the current timestep. Additional iterations are used only when the residual becomes larger than a specified threshold value. Numerical results show that only a few iterations on average are necessary to improve the MSFV results significantly, even for very challenging problems. Therefore, the proposed adaptive strategy yields efficient and accurate simulation of multiphase flow in heterogeneous porous media.

Interaccion among systems of finite size in predictive relativistic mechanics I. General framework

We derive a Hamiltonian formulation for the three-dimensional formalism of predictive relativistic mechanics. This Hamiltonian structure is used to derive a set of dynamical equations describing the interaction among systems in perturbation theory.

Classical predictive electrodynamics of two charges with radiation: General framework. I.

Outgoing radiation is introduced in the framework of the classical predictive electrodynamics using LorentzDiracs equation as a subsidiary condition. In a perturbative scheme in the charges the first radiative self-terms of the accelerations, momentum and angular momentum of a two charge system without external field are calculated.