Towards higher predictability in enzyme engineering : investigation of protein epistasis in dynamic ß-lactamases and Cal-A lipase

L'ingénierie enzymatique est un outil très avantageux dans l'industrie biotechnologique. Elle permet d'adapter les enzymes à une activité ou à une condition de réaction spécifique. En outre, elle peut permettre de déchiffrer les éléments clés qui ont facilité leur modification. Bien que l'ingénierie enzymatique soit largement pratiquée, elle comporte encore plusieurs goulets d'étranglement. Certains de ces goulets d'étranglement sont techniques, comme le développement de méthodologies pour la création de banques de mutations ciblées ou la réalisation de criblages à haut débit, et d'autres sont conceptuels, comme le déchiffrage des caractéristiques clés pertinentes d'une protéine cible pour la réussite d'un projet d'ingénierie. Parmi ces défis, l'épistasie intra-génique, ou la non-additivité des effets phénotypiques des mutations, est une caractéristique qui entrave grandement la prévisibilité. L'amélioration de l'ingénierie enzymatique nécessite une approche multidisciplinaire qui inclut une meilleure compréhension des relations structure-fonction-évolution. Cette thèse vise à contribuer à l'avancement de l'ingénierie enzymatique en étudiant deux systèmes modèles. Premièrement, des variantes dynamiques de la ß-lactamase TEM-1 ont été choisies pour étudier le lien entre la dynamique des protéines et l'évolution. La ß-lactamase TEM-1 a été largement caractérisée dans la littérature, ce qui s'est traduit par des connaissances approfondies sur son mécanisme de réaction, ses caractéristiques structurelles et son évolution. Les variantes de la ß-lactamase TEM-1 utilisées comme système modèle dans cette thèse ont été largement caractérisées, montrant une dynamique accrue à l'échelle temporelle pertinente pour la catalyse (µs à ms) mais maintenant la reconnaissance du substrat. Dans cette thèse, l'évolution in vitro de ces variantes dynamiques a été réalisée par des cycles itératifs de mutagenèse et de sélection aléatoires pour permettre une exploration impartiale du paysage de ‘fitness’. Nous démontrons que la présence de ces mouvements particuliers au début de l'évolution a permis d'accéder à des voies de mutations connues. De plus, des interactions épistatiques connues ont été introduites dans les variantes dynamiques. Leur caractérisation in silico et cinétique a révélé que les mouvements supplémentaires sur l'échelle de temps de la catalyse ont permis d'accéder à des conformations conduisant à une fonction améliorée, comme dans le TEM-1 natif. Dans l'ensemble, nous démontrons que l'évolution de la b-lactamase TEM-1 vers une nouvelle fonction est compatible avec divers mouvements à l'échelle de temps µs à ms. Il reste à savoir si cela peut se traduire par d'autres enzymes ayant un potentiel biotechnologique. Deuxièmement, la lipase Cal-A, pertinente sur le plan industriel, a été choisie pour identifier les caractéristiques qui pourraient faciliter son ingénierie. La lipase Cal-A présente des caractéristiques telles que la polyvalence du substrat et une grande stabilité thermique et réactivité qui la rendent attrayante pour la modification des triglycérides ou la synthèse de molécules pertinentes dans les industries alimentaire et pharmaceutique. Contrairement à TEM-1, la plupart des études d'évolution in vitro de la lipase Cal-A ont été réalisées dans un but industriel, avec une exploration limitée de l'espace de mutation. Par conséquent, les caractéristiques qui définissent la fonction de la lipase Cal-A restent insaisissables. Dans cette thèse, nous faisons état de la mutagenèse ciblée de la lipase Cal-A, confirmant l'existence d'une région clé pour la reconnaissance du substrat. Cela a été fait en combinant une nouvelle méthodologie de création de bibliothèque basée sur l'assemblage Golden-gate avec une visualisation structurelle basée sur des scripts pour identifier et cartographier les mutations sélectionnées dans la structure 3D. La caractérisation et la déconvolution de deux des plus aptes ont révélé l'existence d'une épistasie dans l'évolution de la lipase Cal-A vers une nouvelle fonction. Dans l'ensemble, nous démontrons que l’identification d'une variété de propriétés suite à la mutagenèse ciblée peut grandement améliorer la connaissance d'une enzyme. Cette information peut être appliquée pour améliorer l'efficacité de l'ingénierie dirigée.Enzyme engineering is a tool with great utility in the biotechnological industry. It allows to tailor enzymes to a specific activity or reaction condition. In addition, it can allow to decipher key elements that facilitated their modification. While enzyme engineering is extensively practised, it still entails several bottlenecks. Some of these bottlenecks are technical such as the development of methodologies for creating targeted mutational libraries or performing high-throughput screening and some are conceptual such as deciphering the key relevant features in a target protein for a successful engineering project. Among these challenges, intragenic epistasis, or the non-additivity of the phenotypic effects of mutations, is a feature that greatly hinders predictability. Improving enzyme engineering needs a multidisciplinary approach that includes gaining a better understanding of structure-function-evolution relations. This thesis seeks to contribute in the advancement of enzyme engineering by investigating two model systems. First, dynamic variants of TEM-1 ß-lactamase were chosen to investigate the link between protein dynamics and evolution. TEM-1 ß-lactamase has been extensively characterized in the literature, which has translated into extensive knowledge on its reaction mechanism, structural features and evolution. The variants of TEM-1 ß-lactamase used as model system in this thesis had been extensively characterized, showing increased dynamics at the timescale relevant to catalysis (µs to ms) but maintaining substrate recognition. In this thesis, in vitro evolution of these dynamic variants was done by iterative rounds of random mutagenesis and selection to allow an unbiased exploration of the fitness landscape. We demonstrate that the presence of these particular motions at the outset of evolution allowed access to known mutational pathways. In addition, known epistatic interactions were introduced in the dynamic variants. Their in silico and kinetic characterization revealed that the additional motions on the timescale of catalysis allowed access to conformations leading to enhanced function, as in native TEM-1. Overall, we demonstrate that the evolution of TEM-1 b-lactamase toward new function is compatible with diverse motions at the µs to ms timescale. Whether this can be translated to other enzymes with biotechnological potential remains to be explored. Secondly, the industrially relevant Cal-A lipase was chosen to identify features that could facilitate its engineering. Cal-A lipase presents characteristics such as substrate versatility and high thermal stability and reactivity that make it attractive for modification of triglycerides or synthesis of relevant molecules in the food and pharmaceutical industries. Contrary to TEM-1, most in vitro evolution studies of Cal-A lipase have been done towards an industrially-specified goal, with limited exploration of mutational space. As a result, features that define function in Cal-A lipase remain elusive. In this thesis, we report on focused mutagenesis of Cal-A lipase, confirming the existence of a key region for substrate recognition. This was done by combining a novel library creation methodology based on Golden-gate assembly with script-based structural visualization to identify and map the selected mutations into the 3D structure. The characterization and deconvolution of two of the fittest revealed the existence of epistasis in the evolution of Cal-A lipase towards new function. Overall, we demonstrate that mapping a variety of properties following mutagenesis targeted to specific regions can greatly improve knowledge of an enzyme that can be applied to improve the efficiency of directed engineering

DANCE AND CHOREOGRAPHY COMPETENCE OF UNIVERSITY PHYSICAL EDUCATION TEACHERS

The research determined the Physical Education (PE) Teachers’ dance and choreography competence among government owned higher education institutions (HEIs) in Eastern Visayas Region, Philippines. These competences include subject matter, pedagogy, assessment, guidance, classroom management for dance competence, dance techniques, leadership, creativity and discipline on choreography competence. The study also examined the degree of association between the level of dance and choreography competences among PE Teachers, and investigated the problems encountered by the PE Teachers in doing dance choreography. A descriptive survey research design was used as the main data gathering tool. A Focus Group Discussion (FGD) was also conducted to triangulate the validity and reliability of the generated information. The study was implemented in ten HEIs with forty university PE Teachers as research respondents. Findings revealed that PE Teachers in the study area are very competent in dance. They know what they are teaching, know how to teach, evaluate, guide students and has classroom management. The same hold true for PE Teachers dance choreography competence. They can discipline their students and know the dance technique of the dance type they teach. They possess the knowledge and the skills along the courses they teach. The finding also indicated a significant direct relationship between dance and choreography competences among tertiary PE Teachers. The most common problem encountered by PE Teachers in doing choreography is on the attitude of the dancers while least common problem pertains to the age of the students where older students are hesitant than the younger ones when told to perform. Notwithstanding the various encountered problems, PE Teachers are still very competent and resourceful with lots of initiatives on how to deliver their tasks in teaching dance choreography. They possess love and passion toward their jobs and willing to pursue what needs to be accomplished.  Article visualizations

A study of the communication cost of the FFT on torus multicomputers

The computation of a one-dimensional FFT on a c-dimensional torus multicomputer is analyzed. Different approaches are proposed which differ in the way they use the interconnection network. The first approach is based on the multidimensional index mapping technique for the FFT computation. The second approach starts from a hypercube algorithm and then embeds the hypercube onto the torus. The third approach reduces the communication cost of the hypercube algorithm by pipelining the communication operations. A novel methodology to pipeline the communication operations on a torus is proposed. Analytical models are presented to compare the different approaches. This comparison study shows that the best approach depends on the number of dimensions of the torus and the communication start-up and transfer times. The analytical models allow us to select the most efficient approach for the available machine.Peer ReviewedPostprint (published version

Voluntariat per a la promoció de l’activitat física i cultural en les persones majors

Los principales contenidos tratados se centran en la importancia de la actividad física y la necesidad de cambiar los hábitos, así como en la actitud personal hacia la actividad física y el mantenimiento. Se plantean las consecuencias derivadas de la actividad física limitada en la vida de las personas mayores y se destaca que la actividad física regular cumple una función muy importante en el mantenimiento y que un determinado nivel de actividad y flexibilidad permite disfrutar de la movilidad básica en el día a día a las personas mayores

Hypercube algorithms on mesh connected multicomputers

A new methodology named CALMANT (CC-cube Algorithms on Meshes and Tori) for mapping a type of algorithm that we call CC-cube algorithm onto multicomputers with hypercube, mesh, or torus interconnection topology is proposed. This methodology is suitable when the initial problem can be expressed as a set of processes that communicate through a hypercube topology (a CC-cube algorithm). There are many important algorithms that fit into the CC-cube type. CALMANT is based on three different techniques: (a) the standard embedding to assign the processes of the algorithm to the nodes of the mesh multicomputer; (b) the communication pipelining technique to increase the level of communication parallelism inherent in the CC-cube algorithms; and (c) optimal message-scheduling algorithms proposed in this work in order to avoid conflicts and minimizing in this way the communication time. Although CALMANT is proposed for multicomputers with different interconnection network topologies, the paper only focuses on the particular case of meshes.Peer ReviewedPostprint (published version

‘Risking enchantment’: how are we to view the smoking person?

The idea of the smoking person portrayed in public health has been criticised as being based on too narrow a view of human nature. This article discusses that view: that of a person with a stable core and epiphenomenal ‘behaviours’ that can be removed by rational persuasion or Pavlovian manipulation, and examines social scientific critiques of it. The social sciences explore the meanings smoking has for individuals and portray human identity as malleable, the result of ongoing interactions with human and non-human others. Aspects of smokers’ experience revealed in qualitative research – descriptions of cigarettes as ‘companions’ or ‘friends’, deep reliance, sensual pleasure – are sometimes difficult to articulate but can be given full voice in the context of the literary arts. We explore some examples of these sources and argue that a complete picture of smoking meanings is impossible without reference to them. We take a pragmatic approach, following the philosopher William James, who argued that emotional and spiritual experiences contribute to the truth of human existence as much as material explanations, to suggest that this understanding should be part of a critical but supportive engagement with public health research in order to develop more nuanced and humane approaches to smoking cessation

La universidad con condiciones

El modelo de universidad nacional está siendo sustituido por el de universidad global. A la vez se imponen nuevos dogmas bajo rótulos económicos, mientras se operan, al parecer espontáneamente, cambios culturales importantes. La universidad española fue una de las instancias que el régimen post-franquista cuidó; ahora ese régimen se siente fuerte frente a ella y por otra parte esa universidad -como toda la estructura productiva- requiere una reforma. No está previsto que esa reforma vaya exactamente en el sentido de una mayor presencia social, sino empresarial, ni en el de una mayor democracia interna, sino de un mayor disciplinamiento. Toda resistencia o alternativa debe tener en cuenta la nueva situación y hacer frente común con fuerzas aún dispersas, pero de capacidad constituyente.The model of the national university is being substituted by that of the global university. At the same time, new dogmas that appear with economic labels are being imposed. All this is accompanied by important cultural changes. The Spanish university was one of the instances that was taken care of by the post-Francoist regime. Now, this regime feels strong against it, and on the other hand this university - as the whole of the productive structure - requires a reform. It is not expected that this reform will go in the direction of the university having more social presence, but rather more presence of companies. It is neither expected that it will imply more internal democracy, but more discipline. All resistance or alternative has to take into account the new situation and join the still dispersed forces that still have, however, a constitutive capacity