Design and synthesis of short antimicrobial peptides for plant protection. Study of their mode of action

Aquesta tesi doctoral està basada en el desenvolupament de nous agents antimicrobians derivats del pèptid híbrid cecropina A-melitina WKLFKKILKVL-NH2 (Pep3) que siguin sostenibles i útils per al control de malalties de plantes. Es van dissenyar i sintetitzar més de 133 anàlegs de Pep3 mitjançant química combinatòria. Es van obtenir anàlegs de Pep3 amb una elevada activitat contra fitopatògens i que presentaven baixa toxicitat. Els millors anàlegs van presentar eficàcies comparables amb pesticides de referència en la prevenció d'infeccions causades per fitopatògens. Es va estudiar el mecanisme d'acció de KKLFKKILKYL-NH2 (BP100) investigant la seva interacció amb models de membrana mitjançant tècniques espectroscòpiques. Es va observar la capacitat de BP100 a induir la permeabilització, la neutralització, i l'agregació de vesícules lipídiques aniòniques a una determinada concentració llindar. Es va deduir una equació que relaciona la CMI d'un pèptid antimicrobià amb la constant de partició i la concentració llindar en la membrana.

Parameter estimation for a phenomenological model of the cardiac action potential

The action potential (ap) of a cardiac cell is made up of a complex balance of ionic currents which flow across the cell membrane in response to electrical excitation of the cell. Biophysically detailed mathematical models of the ap have grown larger in terms of the variables and parameters required to model new findings in subcellular ionic mechanisms. The fitting of parameters to such models has seen a large degree of parameter and module re-use from earlier models. An alternative method for modelling electrically exciteable cardiac tissue is a phenomenological model, which reconstructs tissue level ap wave behaviour without subcellular details. A new parameter estimation technique to fit the morphology of the ap in a four variable phenomenological model is presented. An approximation of a nonlinear ordinary differential equation model is established that corresponds to the given phenomenological model of the cardiac ap. The parameter estimation problem is converted into a minimisation problem for the unknown parameters. A modified hybrid Nelder–Mead simplex search and particle swarm optimization is then used to solve the minimisation problem for the unknown parameters. The successful fitting of data generated from a well known biophysically detailed model is demonstrated. A successful fit to an experimental ap recording that contains both noise and experimental artefacts is also produced. The parameter estimation method’s ability to fit a complex morphology to a model with substantially more parameters than previously used is established.

Bustle behind the scenes of action : nemosis as a model for fibroblast inflammatory activation

Wound healing is a complex process that requires an interplay between several cell types. Classically, fibroblasts have been viewed as producers of extracellular matrix, but more recently they have been recognized as orchestrators of the healing response, promoting and directing, inflammation and neovascularization processes. Compared to those from healthy tissue, inflammation-associated fibroblasts display a dramatically altered phenotype and have been described as sentinel cells, able to switch to an immunoregulatory profile on cue. However, the activation mechanism still remains largely uncharacterized. Nemosis is a model for stromal fibroblast activation. When normal human primary fibroblasts are deprived of growth support they cluster, forming multicellular spheroids. Clustering results in upregulation of proinflammatory markers such as cyclooxygenase-2 and secretion of prostaglandins, proteinases, cytokines, and growth factors. Fibroblasts in nemosis induce wound healing and tumorigenic responses in many cell types found in inflammatory and tumor microenvironments. This study investigated the effect of nemotic fibroblasts on two components of the vascular system, leukocytes and endothelium, and characterized the inflammation-promoting responses that arose in these cell types. Fibroblasts in nemosis were found to secrete an array of chemotactic cytokines and attract leukocytes, as well as promote their adhesion to the endothelium. Nuclear factor-kB, the master regulator of many inflammatory responses, is activated in nemotic fibroblasts. Nemotic fibroblasts are known to produce large amounts of hepatocyte growth factor, a motogenic and angiogenic factor. Also, as shown in this study, they produce vascular endothelial growth factor. These two factors induced migratory and sprouting responses in endothelial cells, both required for neovascularization. Nemotic fibroblasts also caused a decrease in the expression of adherens and tight junction components on the surface of endothelial cells. The results allow the conclusion that fibroblasts in nemosis share many similarities with inflammation-associated fibroblasts. Both inflammation and stromal fibroblasts are known to be involved in tumorigenesis and tumor progression. Nemosis may be viewed as a model for stromal fibroblast activation, or it may correlate with cell-cell interactions between adjacent fibroblasts in vivo. Nevertheless, due to nemosis-derived production of proinflammatory cytokines and growth factors, fibroblast nemosis may have therapeutic potential as an inducer of controlled tissue repair. Knowledge of stromal fibroblast activation gained through studies of nemosis, could provide new strategies to control unwanted inflammation and tumor progression.

Disruption of Saccharomyces cerevisiae by Plantaricin 149 and investigation of its mechanism of action with biomembrane model systems

The action of a synthetic antimicrobial peptide analog of Plantaricin 149 (Pln149a) against Saccharomyces cerevisiae and its interaction with biomembrane model systems were investigated. Pln149a was shown to inhibit S. cerevisiae growth by more than 80% in YPD medium, causing morphological changes in the yeast wall and remaining active and resistant to the yeast proteases even after 24 h of incubation. Different membrane model systems and carbohydrates were employed to better describe the Pln149a interaction with cellular components using circular dichroism and fluorescence spectroscopies, adsorption kinetics and surface elasticity in Langmuir monolayers. These assays showed that Pln149a does not interact with either mono/polysaccharides or zwitterionic LUVs, but is strongly adsorbed to and incorporated into negatively charged surfaces, causing a conformational change in its secondary structure from random-coil to helix upon adsorption. From the concurrent analysis of Pln149a adsorption kinetics and dilatational surface elasticity data, we determined that 2.5 mu M is the critical concentration at which Pln149a will disrupt a negative DPPG monolayer. Furthermore, Pln149a exhibited a carpet-like mechanism of action, in which the peptide initially binds to the membrane, covering its surface and acquiring a helical structure that remains associated to the negatively charged phospholipids. After this electrostatic interaction, another peptide region causes a strain in the membrane, promoting its disruption. (C) 2009 Elsevier B.V. All rights reserved.

The calcium wave model of the perception-action cycle: Evidence from semantic relevance in memory experiments

We present a general model of brain function (the calcium wave model), distinguishing three processing modes in the perception-action cycle. The model provides an interpretation of the data from experiments on semantic memory conducted by the authors. © 2013 Pereira Jr, Santos and Barros.

'Defending the rights'. An integrated model of collective action for "public water" in Italy

In many communities, supplying water for the people is a huge task and the fact that this essential service can be carried out by the private sector respecting the right to water, is a debated issue. This dissertation investigates the mechanisms through which a 'perceived rights violation' - which represents a specific form of perceived injustice which derives from the violation of absolute moral principles – can promote collective action. Indeed, literature on morality and collective action suggests that even if many people apparently sustain high moral principles (like human rights), only a minority decides to act in order to defend them. Taking advantage of the political situation in Italy, and the recent mobilization for "public water" we hypothesized that, because of its "sacred value", the perceived violation of the right to water facilitates identification with the social movement and activism. Through five studies adopting qualitative and quantitative methods, we confirmed our hypotheses demonstrating that the perceived violation of the right to water can sustain activism and it can influence vote intentions at the referendum for 'public water'. This path to collective action coexists with other 'classical' predictors of collective action, like instrumental factors (personal advantages, efficacy beliefs) and anger. The perceived rights violation can derive both from personal values (i.e. universalism) and external factors (i.e. a mobilization campaign). Furthermore, we demonstrated that it is possible to enhance the perceived violation of the right to water and anger through a specifically designed communication campaign. The final chapter summarizes the main findings and discusses the results, suggesting some innovative line of research for collective action literature.

Early detection of action potential duration alternans using an autoregressive model

Annual Meeting of the Biophysical Society, San Diego, USA

Using an adaptive autoregressive model for the early detection of action potential duration alternans

Cardiostim 2012, Nice, France

Motivation in action: A process model of L2 motivation

As part of a long-term project aimed at designing classroom interventions to motivate language learners, we have searched for a motivation model that could serve as a theoretical basis for the methodological applications. We have found that none of the existing models we considered were entirely adequate for our purpose for three reasons: (1) they did not provide a sufficiently comprehensive and detailed summary of all the relevant motivational influences on classroom behaviour; (2) they tended to focus on how and why people choose certain courses of action, while ignoring or playing down the importance of motivational sources of executing goal-directed behaviour; and (3) they did not do justice to the fact that motivation is not static but dynamically evolving and changing in time, making it necessary for motivation constructs to contain a featured temporal axis. Consequently, partly inspired by Heckhausen and Kuhl's 'Action Control Theory', we have developed a new 'Process Model of L2 Motivation', which is intended both to account for the dynamics of motivational change in time and to synthesise many of the most important motivational conceptualisations to date. In this paper we describe the main components of this model, also listing a number of its limitations which need to be resolved in future research.

Generalised self-efficacy in relation to the life transitions of adult learners in a university setting : towards a narrative constructivist model of self-regulation

This paper demonstrates a model of self-regulation based on a qualitative research project with adult learners undertaking an undergraduate degree. The narrative about the participant’s life transitions, co-constructed with the researcher, yielded data about their generalised self-efficacy and resulted in a unique self-efficacy narrative for each participant. A model of self-regulation is proposed with potential applications for coaching, counselling and psychotherapy. A narrative method was employed to construct narratives about an individual’s self-efficacy in relation to their experience of learning and life transitions. The method involved a cyclical and iterative process using qualitative interviews to collect life history data from participants. In addition, research participants completed reflective homework tasks, and this data was included in the participant’s narratives. A highly collaborative method entailed narratives being co-constructed by researcher and research participants as the participants were guided in reflecting on their experience in relation to learning and life transitions; the reflection focused on behaviour, cognitions and emotions that constitute a sense of self-efficacy. The analytic process used was narrative analysis, in which life is viewed as constructed and experienced through the telling and retelling of stories and hence the analysis is the creation of a coherent and resonant story. The method of constructing self-efficacy narratives was applied to a sample of mature aged students starting an undergraduate degree. The research outcomes confirmed a three-factor model of self-efficacy, comprising three interrelated stages: initiating action, applying effort, and persistence in overcoming difficulties. Evaluation of the research process by participants suggested that they had gained an enhanced understanding of self-efficacy from their participation in the research process, and would be able to apply this understanding to their studies and other endeavours in the future. A model of self-regulation is proposed as a means for coaches, counsellors and psychotherapists working from a narrative constructivist perspective to assist clients facing life transitions by helping them generate selfefficacious cognitions, emotions and behaviour.

Testing a model of physical activity among mothers and fathers of young children: integrating self-determined motivation, planning, and the theory of planned behavior

Parents are at risk for inactivity; however, research into understanding parental physical activity (PA) is scarce. We integrated self-determined motivation, planning, and the theory of planned behavior (TPB) to better understand parental PA. Parents (252 mothers, 206 fathers) completed a main questionnaire assessing measures underpinning these constructs and a 1-week follow-up of PA behavior to examine whether self-determined motivation indirectly influenced intention via the TPB variables (i.e., attitude, subjective norm, and perceived behavioral control) and intention indirectly influenced behavior via planning. We found self-determined motivation on intention was fully mediated by the TPB variables and intention on behavior was partially mediated by the planning variables. In addition, slight differences in the model’s paths between the sexes were revealed. The results illustrate the range of important determinants of parental PA and provide support for the integrated model in explaining PA decision making as well as the importance of examining sex differences.