Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability

We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to −1.49 (more stable N239Y) kcal mol−1, respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancer-associated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN268D), which is stabilized by 2.65 kcal mol−1 and Tm raised by 5.6°C is of potential interest for trials in vivo.

Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes

Design of hydroxyproline (Hyp)-rich glycoproteins (HRGPs) offers an approach for the structural and functional analysis of these wall components, which are broadly implicated in plant growth and development. HRGPs consist of multiple small repetitive “glycomodules” extensively O-glycosylated through the Hyp residues. The patterns of Hyp-O-glycosylation are putatively coded by the primary sequence as described by the Hyp contiguity hypothesis, which predicts contiguous Hyp residues to be attachment sites of small arabinooligosaccharides (1–5 Ara residues/Hyp); while clustered, noncontiguous Hyp residues are sites of arabinogalactan polysaccharide attachment. As a test, we designed two simple HRGPs as fusion proteins with green fluorescent protein. The first was a repetitive Ser-Hyp motif that encoded only clustered noncontiguous Hyp residues, predicted polysaccharide addition sites. The resulting glycoprotein had arabinogalactan polysaccharide O-linked to all Hyp residues. The second construct, based on the consensus sequence of a gum arabic HRGP, contained both arabinogalactan and arabinooligosaccharide addition sites and, as predicted, gave a product that contained both saccharide types. These results identify an O-glycosylation code of plants.

Deciphering the design of the tropomyosin molecule

The crystal structure at 2.0-Å resolution of an 81-residue N-terminal fragment of muscle α-tropomyosin reveals a parallel two-stranded α-helical coiled-coil structure with a remarkable core. The high alanine content of the molecule is clustered into short regions where the local 2-fold symmetry is broken by a small (≈1.2-Å) axial staggering of the helices. The joining of these regions with neighboring segments, where the helices are in axial register, gives rise to specific bends in the molecular axis. We observe such bends to be widely distributed in two-stranded α-helical coiled-coil proteins. This asymmetric design in a dimer of identical (or highly similar) sequences allows the tropomyosin molecule to adopt multiple bent conformations. The seven alanine clusters in the core of the complete molecule (which spans seven monomers of the actin helix) promote the semiflexible winding of the tropomyosin filament necessary for its regulatory role in muscle contraction.

A test of the "jigsaw puzzle" model for protein folding by multiple methionine substitutions within the core of T4 lysozyme.

To test whether the structure of a protein is determined in a manner akin to the assembly of a jigsaw puzzle, up to 10 adjacent residues within the core of T4 lysozyme were replaced by methionine. Such variants are active and fold cooperatively with progressively reduced stability. The structure of a seven-methionine variant has been shown, crystallographically, to be similar to wild type and to maintain a well ordered core. The interaction between the core residues is, therefore, not strictly comparable with the precise spatial complementarity of the pieces of a jigsaw puzzle. Rather, a certain amount of give and take in forming the core structure is permitted. A simplified hydrophobic core sequence, imposed without genetic selection or computer-based design, is sufficient to retain native properties in a globular protein.

Review and extension of the McCabe-Thiele method covering multiple feeds, products and heat transfer stages

The McCabe-Thiele method is a classical approximate graphical method for the conceptual design of binary distillation columns which is still widely used, mainly for didactical purposes, though it is also valuable for quick preliminary calculations. Nevertheless, no complete description of the method has been found and situations such as different thermal feed conditions, multiple feeds, possibilities to extract by-products or to add or remove heat, are not always considered. In the present work we provide a systematic analysis of such situations by developing the generalized equations for: a) the operating lines (OL) of each sector, and b) the changeover line that provides the connection between two consecutive trays of the corresponding sectors separated by a lateral stream of feed, product, or a heat removal or addition.

Some examples of modeling in chemical engineering: thermal treatment of polymers, phase equilibrium calculations and process design

Presentation submitted to PSE Seminar, Chemical Engineering Department, Center for Advanced Process Design-making (CAPD), Carnegie Mellon University, Pittsburgh (USA), October 2012.

Mathematical programming model for heat exchanger design through optimization of partial objectives

Mathematical programming can be used for the optimal design of shell-and-tube heat exchangers (STHEs). This paper proposes a mixed integer non-linear programming (MINLP) model for the design of STHEs, following rigorously the standards of the Tubular Exchanger Manufacturers Association (TEMA). Bell–Delaware Method is used for the shell-side calculations. This approach produces a large and non-convex model that cannot be solved to global optimality with the current state of the art solvers. Notwithstanding, it is proposed to perform a sequential optimization approach of partial objective targets through the division of the problem into sets of related equations that are easier to solve. For each one of these problems a heuristic objective function is selected based on the physical behavior of the problem. The global optimal solution of the original problem cannot be ensured even in the case in which each of the sub-problems is solved to global optimality, but at least a very good solution is always guaranteed. Three cases extracted from the literature were studied. The results showed that in all cases the values obtained using the proposed MINLP model containing multiple objective functions improved the values presented in the literature.

Analysis of the Connecting Zone between Consecutive Sections in Distillation Columns Covering Multiple Feeds, Products and Heat Transfer Stages

In the present work, we provide a systematic analysis about all tine streams involved in the zone connecting two consecutive sections for the design of distillation columns with different thermal feed conditions, product extractions and heat additions or withdrawals. This analysis allows a better understanding of what happens on a feed or side draw (of mass or energy) stage, what compositions are or are not in equilibrium, and the impact on internal liquid and vapor flows.

3D teaching: the ideal complement for professionals in design and construction

Today, the requirement of professional skills to university students is constantly increasing in our society. In our opinion, the content offered in official degrees need to be nourished with different variables, enriching their global professional knowledge in a parallel way; that is why, in recent years, there is a great multiplicity of complementary courses at university. One of the most socially demanded technical requirements within the architectural, design or engineering field is the management of 3D drawing software, becoming an indispensable reality in these sectors. Thus, this specific training becomes essential over two-dimension traditional design, because the inclusion of great possibilities of spatial development that go beyond conventional orthographic projections (plans, sections or elevations), allowing modelling and rotation of the selected items from multiple angles and perspectives. Therefore, this paper analyzes the teaching methodology of a complementary course for those technicians in the construction industry interested in computer-aided design, using modelling (SketchupMake) and rendering programs (Kerkythea). The course is developed from the technician point of view, by learning computer management and its application to professional development from a more general to a more specific view through practical examples. The proposed methodology is based on the development of real examples in different professional environments such as rehabilitation, new constructions, opening projects or architectural design. This multidisciplinary contribution improves criticism of students in different areas, encouraging new learning strategies and the independent development of three-dimensional solutions. Thus, the practical implementation of new situations, even suggested by the students themselves, ensures active participation, saving time during the design process and the increase of effectiveness when generating elements which may be represented, moved or virtually tested. In conclusion, this teaching-learning methodology improves the skills and competencies of students to face the growing professional demands of society. After finishing the course, technicians not only improved their expertise in the field of drawing but they also enhanced their capacity for spatial vision; both essential qualities in these sectors that can be applied to their professional development with great success.

Supplementary Material: Analysis of the Connecting Zone Between Consecutive Sections in Distillation Columns Covering Multiple Feeds, Products and Heat Transfer Stages

Supplementary Material: J.A. REYES-LABARTA, M.D. SERRANO and A. MARCILLA. ANALYSIS OF THE CONNECTING ZONE BETWEEN CONSECUTIVE SECTIONS IN DISTILLATION COLUMNS COVERING MULTIPLE FEEDS, PRODUCTS AND HEAT TRANSFER STAGES. Latin American Applied Research an International Journal of Chemical Engineering. 2014, vol. 44(4), 307-312 (http://www.laar.uns.edu.ar/indexes/artic_v4404/44_04_307.pdf)

Optimization of test and maintenance of ageing components consisting of multiple items and addressing effectiveness

There are many models in the literature that have been proposed in the last decades aimed at assessing the reliability, availability and maintainability (RAM) of safety equipment, many of them with a focus on their use to assess the risk level of a technological system or to search for appropriate design and/or surveillance and maintenance policies in order to assure that an optimum level of RAM of safety systems is kept during all the plant operational life. This paper proposes a new approach for RAM modelling that accounts for equipment ageing and maintenance and testing effectiveness of equipment consisting of multiple items in an integrated manner. This model is then used to perform the simultaneous optimization of testing and maintenance for ageing equipment consisting of multiple items. An example of application is provided, which considers a simplified High Pressure Injection System (HPIS) of a typical Power Water Reactor (PWR). Basically, this system consists of motor driven pumps (MDP) and motor operated valves (MOV), where both types of components consists of two items each. These components present different failure and cause modes and behaviours, and they also undertake complex test and maintenance activities depending on the item involved. The results of the example of application demonstrate that the optimization algorithm provide the best solutions when the optimization problem is formulated and solved considering full flexibility in the implementation of testing and maintenance activities taking part of such an integrated RAM model.

Towards a new design of South American integration? Jean Monnet/Robert Schuman Paper Series, Special Issue January 2013

The design of South American integration is becoming different. This has been quite common in the trajectory of over six decades of initiatives aimed at generating institutional frameworks to facilitate regional integration. However, even when it has become apparent that the previous design is undergoing a new process of change, it would be difficult to predict for how long the one that is beginning to take shape will remain in effect. The experience of recent decades suggests great caution in forecasts that are optimistic about any eventual longevity. Several factors are contributing to this redesign. Some are external to the region while others are endogenous. The combination of these factors will influence the future design of South American integration. If past lessons are correctly capitalized and certain advantage is derived from the leeway provided by a decentralized international system with multiple options, we can anticipate that what will predominate in the region will be multidimensional integration agreements (with political and economic objectives at the same time) and with cross-memberships and commitments. If this were the case, the actual impact on regional governance, social and productive integration and the competitive insertion at a global scale will depend largely on the following factors: the quality and sustainability of the strategy for development and global and regional insertion of each country; the combination of a reasonable degree of flexibility and predictability in the commitments made and their corresponding ground rule, and the density of the network of cross-interests that can be achieved as a result of the respective regional integration agreements, reflected in multiple transnational social and production networks.

Design and synthesis of constrained azacyclic pyrrolidine analogues of FTY720 as anticancer agents & metal coordination-controlled and bifunctional catalysis toward tertiary β-Ketols

Cette thèse se compose en deux parties: Première Partie: La conception et la synthèse d’analogues pyrrolidiniques, utilisés comme agents anticancéreux, dérivés du FTY720. FTY720 est actuellement commercialisé comme médicament (GilenyaTM) pour le traitement de la sclérose en plaques rémittente-récurrente. Il agit comme immunosuppresseur en raison de son effet sur les récepteurs de la sphingosine-1-phosphate. A fortes doses, FTY720 présente un effet antinéoplasique. Cependant, à de telles doses, un des effets secondaires observé est la bradycardie dû à l’activation des récepteurs S1P1 et S1P3. Ceci limite son potentiel d’utilisation lors de chimiothérapie. Nos précédentes études ont montré que des analogues pyrrolidiniques dérivés du FTY720 présentaient une activité anticancéreuse mais aucune sur les récepteurs S1P1 et S1P3. Nous avons soumis l’idée qu’une étude relation structure-activité (SARs) pourrait nous conduire à la découverte de nouveaux agents anti tumoraux. Ainsi, deux séries de composés pyrrolidiniques (O-arylmethyl substitué et C-arylmethyl substitué) ont pu être envisagés et synthétisés (Chapitre 1). Ces analogues ont montré d’excellentes activités cytotoxiques contre diverses cellules cancéreuses humaines (prostate, colon, sein, pancréas et leucémie), plus particulièrement les analogues actifs qui ne peuvent pas être phosphorylés par SphK, présentent un plus grand potentiel pour le traitement du cancer sans effet secondaire comme la bradycardie. Les études mécanistiques suggèrent que ces analogues de déclencheurs de régulation négative sur les transporteurs de nutriments induisent une crise bioénergétique en affamant les cellules cancéreuses. Afin d’approfondir nos connaissances sur les récepteurs cibles, nous avons conçu et synthétisé des sondes diazirine basées sur le marquage d’affinité aux photons (méthode PAL: Photo-Affinity Labeling) (Chapitre 2). En s’appuyant sur la méthode PAL, il est possible de récolter des informations sur les récepteurs cibles à travers l’analyse LC/MS/MS de la protéine. Ces tests sont en cours et les résultats sont prometteurs. Deuxième partie: Coordination métallique et catalyse di fonctionnelle de dérivés β-hydroxy cétones tertiaires. Les réactions de Barbier et de Grignard sont des méthodes classiques pour former des liaisons carbone-carbone, et généralement utilisées pour la préparation d’alcools secondaires et tertiaires. En vue d’améliorer la réaction de Grignard avec le 1-iodobutane dans les conditions « one-pot » de Barbier, nous avons obtenu comme produit majoritaire la β-hydroxy cétone provenant de l’auto aldolisation de la 5-hexen-2-one, plutôt que le produit attendu d’addition de l’alcool (Chapitre 3). La formation inattendue de la β-hydroxy cétone a également été observée en utilisant d’autres dérivés méthyl cétone. Étonnement dans la réaction intramoléculaire d’une tricétone, connue pour former la cétone Hajos-Parrish, le produit majoritaire est rarement la β-hydroxy cétone présentant la fonction alcool en position axiale. Intrigué par ces résultats et après l’étude systématique des conditions de réaction, nous avons développé deux nouvelles méthodes à travers la synthèse sélective et catalytique de β-hydroxy cétones spécifiques par cyclisation intramoléculaire avec des rendements élevés (Chapitre 4). La réaction peut être catalysée soit par une base adaptée et du bromure de lithium comme additif en passant par un état de transition coordonné au lithium, ou bien soit à l’aide d’un catalyseur TBD di fonctionnel, via un état de transition médiée par une coordination bidenté au TBD. Les mécanismes proposés ont été corroborés par calcul DFT. Ces réactions catalytiques ont également été appliquées à d’autres substrats comme les tricétones et les dicétones. Bien que les efforts préliminaires afin d’obtenir une enantioselectivité se sont révélés sans succès, la synthèse et la recherche de nouveaux catalyseurs chiraux sont en cours.

Predictors of Nasal Obstruction: Quantification and Assessment Using Multiple Grading Scales

Objective. To evaluate the association between nasal obstruction and (1) demographic factors, (2) medical history, (3) physical tests, and (4) nasal exam findings. Study Design. CASE SERIES: Methods. Chart review at a tertiary medical center. Results. Two hundred-forty consecutive patients (52.1 ± 17.5 years old, with a Nasal Obstruction Symptom Evaluation (NOSE) score of 32.0 ± 24.1) were included. Demographic factors and inferior turbinate sizes were not associated with NOSE score or Nasal Obstruction Visual Analog Scale (NO-VAS). A significant association was found between higher NOSE score on univariate analysis and positive history of nasal trauma (p = 0.0136), allergic rhinitis (p < 0.0001), use of nasal steroids (p = 0.0108), higher grade of external nasal deformity (p = 0.0149), higher internal nasal septal deviation grade (p = 0.0024), and narrow internal nasal valve angle (p < 0.0001). Multivariate analysis identified the following as independent predictors of high NOSE score: NO-VAS: ≥50 (Odds Ratio (OR) = 17.6 (95% CI 5.83-61.6), p < 0.0001), external nasal deformity: grades 2-4 (OR = 4.63 (95% CI 1.14-19.9), p = 0.0339), and allergic rhinitis: yes (OR = 5.5 (95% CI 1.77-18.7), p = 0.0041). Conclusion. Allergic rhinitis, NO-VAS score ≥ 50, and external nasal deformity (grades 2-4) were statistically significant independent predictors of high NOSE scores on multivariate analysis. Inferior turbinate size was not associated with NOSE scores or NO-VAS.

Design and synthesis of constrained azacyclic pyrrolidine analogues of FTY720 as anticancer agents & metal coordination-controlled and bifunctional catalysis toward tertiary β-Ketols

Cette thèse se compose en deux parties: Première Partie: La conception et la synthèse d’analogues pyrrolidiniques, utilisés comme agents anticancéreux, dérivés du FTY720. FTY720 est actuellement commercialisé comme médicament (GilenyaTM) pour le traitement de la sclérose en plaques rémittente-récurrente. Il agit comme immunosuppresseur en raison de son effet sur les récepteurs de la sphingosine-1-phosphate. A fortes doses, FTY720 présente un effet antinéoplasique. Cependant, à de telles doses, un des effets secondaires observé est la bradycardie dû à l’activation des récepteurs S1P1 et S1P3. Ceci limite son potentiel d’utilisation lors de chimiothérapie. Nos précédentes études ont montré que des analogues pyrrolidiniques dérivés du FTY720 présentaient une activité anticancéreuse mais aucune sur les récepteurs S1P1 et S1P3. Nous avons soumis l’idée qu’une étude relation structure-activité (SARs) pourrait nous conduire à la découverte de nouveaux agents anti tumoraux. Ainsi, deux séries de composés pyrrolidiniques (O-arylmethyl substitué et C-arylmethyl substitué) ont pu être envisagés et synthétisés (Chapitre 1). Ces analogues ont montré d’excellentes activités cytotoxiques contre diverses cellules cancéreuses humaines (prostate, colon, sein, pancréas et leucémie), plus particulièrement les analogues actifs qui ne peuvent pas être phosphorylés par SphK, présentent un plus grand potentiel pour le traitement du cancer sans effet secondaire comme la bradycardie. Les études mécanistiques suggèrent que ces analogues de déclencheurs de régulation négative sur les transporteurs de nutriments induisent une crise bioénergétique en affamant les cellules cancéreuses. Afin d’approfondir nos connaissances sur les récepteurs cibles, nous avons conçu et synthétisé des sondes diazirine basées sur le marquage d’affinité aux photons (méthode PAL: Photo-Affinity Labeling) (Chapitre 2). En s’appuyant sur la méthode PAL, il est possible de récolter des informations sur les récepteurs cibles à travers l’analyse LC/MS/MS de la protéine. Ces tests sont en cours et les résultats sont prometteurs. Deuxième partie: Coordination métallique et catalyse di fonctionnelle de dérivés β-hydroxy cétones tertiaires. Les réactions de Barbier et de Grignard sont des méthodes classiques pour former des liaisons carbone-carbone, et généralement utilisées pour la préparation d’alcools secondaires et tertiaires. En vue d’améliorer la réaction de Grignard avec le 1-iodobutane dans les conditions « one-pot » de Barbier, nous avons obtenu comme produit majoritaire la β-hydroxy cétone provenant de l’auto aldolisation de la 5-hexen-2-one, plutôt que le produit attendu d’addition de l’alcool (Chapitre 3). La formation inattendue de la β-hydroxy cétone a également été observée en utilisant d’autres dérivés méthyl cétone. Étonnement dans la réaction intramoléculaire d’une tricétone, connue pour former la cétone Hajos-Parrish, le produit majoritaire est rarement la β-hydroxy cétone présentant la fonction alcool en position axiale. Intrigué par ces résultats et après l’étude systématique des conditions de réaction, nous avons développé deux nouvelles méthodes à travers la synthèse sélective et catalytique de β-hydroxy cétones spécifiques par cyclisation intramoléculaire avec des rendements élevés (Chapitre 4). La réaction peut être catalysée soit par une base adaptée et du bromure de lithium comme additif en passant par un état de transition coordonné au lithium, ou bien soit à l’aide d’un catalyseur TBD di fonctionnel, via un état de transition médiée par une coordination bidenté au TBD. Les mécanismes proposés ont été corroborés par calcul DFT. Ces réactions catalytiques ont également été appliquées à d’autres substrats comme les tricétones et les dicétones. Bien que les efforts préliminaires afin d’obtenir une enantioselectivité se sont révélés sans succès, la synthèse et la recherche de nouveaux catalyseurs chiraux sont en cours.