Aportación al conocimiento de la brioflora de Andalucía Occidental

Este trabajo es continuación de otros anteriores (Casas y Oliva, 1982 a y b; Oliva, 1987 a y b) en el que seguimos la misma pauta de ellos: recopilación de todas las citas aportadas por los distintos botánicos que con anterioridad han estudiado la zona, e inclusión de nuestros propios hallazgos

AMPs: Rational Design, Synthesis and Biophysical Studies of the Interaction Process with Model Membranes

The emergence of resistance from bacteria to the conventional antibiotics has become a serious global problem during the last years. The onset of resistance is mainly due to the massive and out-of-control use of these drugs in our community. In fact, microorganisms have developed a series of mechanism which render antibiotics ineffective. Therefore, seeking for new anti-infective agents is becoming increasingly necessary. Among these antimicrobial peptides (AMPs) have been proposed. AMPs are a class of peptides with a broad spectrum of activity against different pathogenic organisms (e.g. bacteria, fungi, viruses and cancer cells) and they are part of the innate immune system of virtually all forms of life. Usually, they are composed by 10-50 amino acids and are enriched of positively charged (e.g. lysine and arginine) and hydrophobic residues. Even if the exact action mechanism is still under debate, it is widely accepted that the primary target is represented by the lipid matrix of the pathogens’ membranes. Thus, AMPs interact with them in a non-specific way, inducing membrane destabilization and finally cell death. It is believed that, due to the unique mode of action, AMPs could overcome the problem of resistance to conventional antibiotics in multi-drug resistant bacteria and even to currently used anticancer drugs for the treatment of tumor cells. For these reasons, AMPs have attracted great attention as drugs of the future. Thus, the final goal in studying AMPs is their use as drugs. However, AMPs pharmacological application is not straightforward. Many aspects must be considered. A good AMP should interact selectively with the bacterial membrane, should not be toxic to eukaryotic cells and should be resistant to proteases degradation. To develop AMPs with these improved features it is of fundamental importance to understand the role played by lipid composition and peptide physico-chemical properties in determining peptides activity. To this aim, in my PhD thesis I studied the molecular details, at level of peptide-lipid interaction, of the action mechanism of several antimicrobial peptides by a series of biophysical techniques. In particular, I characterized the interaction of natural amino acids-containing peptide as well as of synthetic peptides composed by unnatural amino acids with model biomembranes. In the first part of the project, I carried out the synthesis of the 9-residue peptide P9Nal(SS) which contains unnatural amino acids. It was designed in order to obtain a peptide with a good antimicrobial activity, low cytotoxicity and high resistance to proteases. Then, biophysical studies of its interaction with eukaryotic and bacterial model membranes were carried out. The obtained information can be very useful in developing antimicrobial agents for biomedical applications. This study is described in detail in the chapter 3. In the chapter 4 is reported the biophysical characterization of the interaction of two P9Nal(SS)-derived peptides, named P9Nal(SR) and P9Trp(SS), with liposomes mimicking bacterial membrane. The two peptides were obtained by replacing some residues in the primary sequence of P9Nal(SS). As shown in chapter 4, the peptides are less hydrophobic than the parent peptide. The obtained data reveal how these substitutions can modulate the membranotropic activity of peptides. In the second part of the project, I faced the problem to understand the molecular details of the action mechanism of (P)GKY20, a natural amino acids-containing peptide modelled on the C-terminus region of the human thrombin. Its good antimicrobial activity and low cytotoxicity is well known. However, its action mechanism has never been studied before. Thus, I performed an extensive biophysical characterization of the interaction of the (P)GKY20 with model membranes. The obtained results elucidated its mechanism of action against bacterial model membrane and, at the same time, allow to understand its low cytotoxicity. All the results concerning this AMP are presented in the chapter 5. The last part of this thesis is devoted to an idea developed during the third year: the encapsulation of AMPs with cyclodextrins (CDs) as a way to protect the peptides from degradation and improve their pharmacological properties. Indeed, antimicrobial peptides containing natural L amino acids are, unfortunately, prone to degradation which limits seriously AMPs applications as drugs. To verify the ability of CDs to encapsulate AMPs without altering their antimicrobial properties, I studied the interaction of (P)GKY20 peptide with sulfobutylether-β-CD and of the obtained complex with bacterial-like liposomes. These preliminary results reveal that (P)GKY20 form a 1:1 stable complex with sulfobutylether-β-CD. Further, the obtained complex is able to perturb the stability of bacterial-like liposomes. Thus, the sulfobutylether CD could represent a suitable encapsulating agent for (P)GKY20 peptide which could improve its pharmacological profile. The obtained results are presented in the chapter 6. Since the peptide interaction with model membranes is quite complex being composed by many steps, a strategy which involves different biophysical techniques was adopted to study a particular aspect of the interaction process. Calorimetric, spectroscopic and microscopic techniques were applied (where possible) and by combining the data, it was possible to depict a possible action mechanism highlighting the key steps. Differential Scanning Calorimetry (DSC) was employed to study the effect of peptide interaction on the liposomes thermotropic properties. Moreover, using an appropriate lipid system, information about lipid self-organization (e.g. lipid domains formation) can be obtained. Steady-State Fluorescence Spectroscopy and Fluorescence Anisotropy were extensively employed to obtain information on both AMPs (e.g. determination of binding constants, degree of insertion into the lipid bilayer) and lipids (e.g. changes in membrane viscosity due to changes in lipid packing). Liposomes morphological changes were monitored by Dynamic Light Scattering (DLS). Instead, changes in peptide secondary structure upon binding were followed by means of Circular Dichroism (CD) and an estimation of secondary structure content was obtained by the spectra deconvolution. Atomic Force Microscopy (AFM) and Confocal Fluorescence Microscopy instruments were employed to directly visualize the effect of peptides on bacterial model membranes

Unraveling the binding characteristics of small ligands to telomeric DNA by pressure modulation

Recently, non-canonical DNA structures, such as G-quadruplexes (GQs), were found to be highly pressure sensitive, suggesting that pressure modulation studies can provide additional mechanistic details of such biomolecular systems. Using FRET and CD spectroscopy as well as binding equilibrium measurements, we investigated the effect of pressure on the binding reaction of the ligand ThT to the quadruplex 22AG in solutions containing different ionic species and a crowding agent mimicking the intracellular milieu. Pressure modulation helped us to identify the different conformational substates adopted by the quadruplex at the different solution conditions and to determine the volumetric changes during complex formation and the conformational transitions involved. The magnitudes of the binding volumes are a hallmark of packing defects and hydrational changes upon ligand binding. The conformational substates of the GQ as well as the binding strength and the stoichiometry of complex formation depend strongly on the solution conditions as well as on pressure. High hydrostatic pressure can also impact GQs inside living cells and thus affect expression of genetic information in deep sea organisms. We show that sub-kbar pressures do not only affect the conformational dynamics and structures of GQs, but also their ligand binding reactions

Influence of University Access Test in the teaching methodology of science teachers

[Resumen] En este artículo se investigan las percepciones del profesorado de ciencias sobre el efecto de las pruebas de acceso a la universidad, en la metodología docente y en las prácticas de evaluación. En el estudio participaron 120 profesores/as con experiencia docente en asignaturas de ciencias de segundo curso de bachillerato. Como instrumentos de recogida de datos se emplearon cuestionarios abiertos y cerrados, así como entrevistas semiestructuradas, combinándose métodos cualitativos y cuantitativos de análisis de datos. En conjunto, se detecta un efecto inhibitorio en el 2º curso de bachillerato de ciertos recursos y metodologías de enseñanza que habitualmente se consideran de interés en el aprendizaje de las ciencias, favoreciéndose en su lugar enfoques de enseñanza tradicionales, con clases teóricas expositivas. A partir de estos resultados se sugiere la necesidad de una reformulación de las pruebas de acceso y la conveniencia de abordar esta problemática dentro de la formación del profesorado[Abstract] In this work, we investigate he perceptions of science teachers about the effect of the University Access Tests in their teaching methodology and their evaluation practices. The study included 120 teachers with teaching experience, in science subjects of the second-year of secondary education. As data collection instruments, open and closed questionnaires were used, as well as semi-structured interviews, combining qualitative and quantitative methods of data analysis. Overall, an inhibitory effect is detected in the 2nd year of the secondary education of certain teaching resources and methodologies that are usually considered of interest in science learning, favoring instead traditional teaching approaches, with expository theoretical classes. Based on these results, we suggest a reformulation of the access tests and the convenience of addressing this problem within teacher training

Influencia de la severidad de maloclusión, calidad de vida y nivel socioeconómico en la autoestima de adolescentes de una institución educativa de la ciudad de Chiclayo, 2019

El presente estudio de investigación tuvo como objetivo determinar la influencia de la severidad de maloclusión, calidad de vida y nivel socioeconómico en la autoestima de adolescentes de una institución educativa de la ciudad de Chiclayo, 2019. Con respecto a la metodología, el estudio tuvo un enfoque cuantitativo, observacional, prospectivo, descriptivo y transversal. La población de estudio estuvo conformada por estudiantes en la etapa de adolescencia media de 3°,4° y 5° grado de secundaria de la institución educativa que cumplieron con los criterios de inclusión y exclusión. Para evaluar las variables del estudio: severidad de maloclusión, calidad de vida, nivel socioeconómico y autoestima se empleó el índice DAI, el cuestionario PIDAQ, la Evaluación de los Niveles Socioeconómicos versión modificada 2011-2012 y la Escala de Autoestima de Rosenberg; respectivamente. Se realizó el análisis de los datos univariado, bivariado y multivariado a través del programa estadístico IBM SPSS Statistics versión 25.0. Al evaluar la influencia de las variables en la autoestima de los adolescentes, a través de la regresión lineal, no se encontró diferencia de los adolescentes con maloclusión normal frente al resto (p=0.392>0.05), tampoco efecto de la calidad de vida (p=0.730>0.05), ni de los adolescentes con nivel socioeconómico marginal (p=0.896) o inferior (p=0.925) frente a los de nivel bajo superior. En conclusión, en el estudio no se encontró significancia estadística que compruebe que existe una influencia de las variables severidad de maloclusión, calidad de vida y nivel socioeconómico en la autoestima

Suppression of Liquid-Liquid Phase Separation and Aggregation of Antibodies by Modest Pressure Application

The high colloidal stability of antibody (immunoglobulin) solutions is important for pharmaceutical applications. Inert cosolutes, excipients, are generally used in therapeutic protein formulations to minimize physical instabilities, such as liquid-liquid phase separation (LLPS), aggregation and precipitation, which are often encountered during manufacturing and storage. Despite their widespread use, a detailed understanding of how excipients modulate the specific protein-protein interactions responsible for these instabilities is still lacking. In this work, we demonstrate the high sensitivity to pressure of globulin condensates as a suitable means to suppress LLPS and subsequent aggregation of concentrated antibody solutions. The addition of excipients has only a minor effect. The high pressure sensitivity observed is due to the fact that these flexible Y-shaped molecules create a considerable amount of void volume in the condensed phase, leading to an overall decrease in the volume of the system upon dissociation of the droplet phase by pressure already at a few tens of to hundred bar. Moreover, we show that immunoglobulin molecules themselves are highly resistant to unfolding under pressure, and can even sustain pressures up to about 6 kbar without conformational changes. This implies that immunoglobulins are resistant to the pressure treatment of foods, such as milk, in high-pressure food-processing technologies, thereby preserving their immunological activity

High pressure treatment promotes the deteriorating effect of cationic antimicrobial peptides on bacterial membranes

The helical structure that cationic antimicrobial peptides (cAMPs) adopt upon interaction with membranes is key to their activity. We show that a high hydrostatic pressure not only increases the propensity of cAMPs to adopt a helical conformation in the presence of bacterial lipid bilayer membranes, but also in bulk solution, and the effect on bacterial membranes persists even up to 10 kbar. Therefore, high-pressure treatment could boost cAMP activity in high-pressure food processing to extend the shelf-life of food

Deep sea osmolytes in action: their effect on protein-ligand binding under high pressure stress

Because organisms living in the deep sea and in the sub-seafloor must be able to cope with hydrostatic pressures up to 1000 bar and more, their biomolecular processes, including ligand-binding reactions, must be adjusted to keep the associated volume changes low in order to function efficiently. Almost all organisms use organic cosolvents (osmolytes) to protect their cells from adverse environmental conditions. They counteract osmotic imbalance, stabilize the structure of proteins and maintain their function. We studied the binding properties of the prototypical ligand proflavine to two serum proteins with different binding pockets, BSA and HSA, in the presence of two prominent osmolytes, trimethylamine-N-oxide (TMAO) and glycine betaine (GB). TMAO and GB play an important role in the regulation and adaptation of life in deep-sea organisms. To this end, pressure dependent fluorescence spectroscopy was applied, supplemented by circular dichroism (CD) spectroscopy and computer modeling studies. The pressure-dependent measurements were also performed to investigate the intimate nature of the complex formation in relation to hydration and packing changes caused by the presence of the osmolytes. We show that TMAO and GB are able to modulate the ligand binding process in specific ways. Depending on the chemical make-up of the protein's binding pocket and thus the thermodynamic forces driving the binding process, there are osmolytes with specific interaction sites and binding strengths with water that are able to mediate efficient ligand binding even under external stress conditions. In the binding of proflavine to BSA and HSA, the addition of both compatible osmolytes leads to an increase in the binding constant upon pressurization, with TMAO being the most efficient, rendering the binding process also insensitive to pressurization even up to 2 kbar as the volume change remains close to zero. This effect can be corroborated by the effects the cosolvents impose on the strength and dynamics of hydration water as well as on the conformational dynamics of the protein

Suppression of liquid-liquid phase separation and aggregation of antibodies by modest pressure application

The high colloidal stability of antibody (immunoglobulin) solutions is important for pharmaceutical applications. Inert cosolutes, excipients, are generally used in therapeutic protein formulations to minimize physical instabilities, such as liquid–liquid phase separation (LLPS), aggregation and precipitation, which are often encountered during manufacturing and storage. Despite their widespread use, a detailed understanding of how excipients modulate the specific protein-protein interactions responsible for these instabilities is still lacking. In this work, we demonstrate the high sensitivity to pressure of globulin condensates as a suitable means to suppress LLPS and subsequent aggregation of concentrated antibody solutions. The addition of excipients has only a minor effect. The high pressure sensitivity observed is due to the fact that these flexible Y-shaped molecules create a considerable amount of void volume in the condensed phase, leading to an overall decrease in the volume of the system upon dissociation of the droplet phase by pressure already at a few tens of to hundred bar. Moreover, we show that immunoglobulin molecules themselves are highly resistant to unfolding under pressure, and can even sustain pressures up to about 6 kbar without conformational changes. This implies that immunoglobulins are resistant to the pressure treatment of foods, such as milk, in high-pressure food-processing technologies, thereby preserving their immunological activity