Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study

Fluorescence spectroscopy andmicroscopy have been utilized as tools in membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Among the latter, 1,6-diphenylhexatriene (DPH) and its trimethylammonium derivative (TMA-DPH) have been extensively used. It is widely believed that, owing to its additional charged group, TMA-DPH is anchored at the lipid/water interface and reports on a bilayer region that is distinct from that of the hydrophobic DPH. In this study, we employ atomistic MD simulations to characterize the behavior of DPH and TMA-DPH in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/cholesterol (4:1) bilayers. We show that although the dynamics of TMA-DPH in thesemembranes is noticeably more hindered than that of DPH, the location of the average fluorophore of TMA-DPH is only ~3–4 Å more shallow than that of DPH. The hindrance observed in the translational and rotational motions of TMA-DPH compared to DPH is mainly not due to significant differences in depth, but to the favorable electrostatic interactions of the former with electronegative lipid atoms instead. By revealing detailed insights on the behavior of these two probes, our results are useful both in the interpretation of past work and in the planning of future experiments using themasmembrane reporters.

Diferenciación molecular de Lemna aequinoctialis y L. Valdiviana (lemnaceae) por patrones proteicos

El estudio se llevó a cabo en el Laboratorio de Genética Molecular de la Universidad Nacional, Heredia, Costa Rica. Se utilizaron plantas acuáticas macrófitas de Lemna valdiviana y L. aequinoctialis previamente identificadas. Estas especies habitan en aguas ricas en sales disueltas y aguas servidas. Recientemente se les consideró como un indicador de la contaminación ocasionada por pesticidas y herbicidas en el agua. La composición proteica llega a niveles entre 38 y 43% de materia seca, importante para diversos usos. La extracción de proteínas totales, por medio de electroforesis discontinua en gel de poliacrilamida, se realizó en condiciones reductoras con SDS (SDS-PAGE). Los geles separadores empleados fueron al 8 y 10%, gel espaciador al 4% y la tinción se efectuó con azul commassie. Los diversos patrones proteicos reflejaron diferencias genéticas entre ambas especies que se pueden deber a las diferencias de hábitat y de reproducción.

Caracterización molecular por patrones electroforéticos de isoenzimas esterasas en Lemna aequinoctialis y Lemna valdiviana (lemnaceae) de Costa Rica (ING)

The Lemnaceae family lacks morphologic and anatomical characteristics with comparative aims and for that reason the molecular studies has been necessary a differentiation. In Costa Rica there are two species of Lemna: Lemna aequinoctialis and Lemna valdiviana. With the objective of a molecular characterization and differentiation of both species were extracted isoenzymes esterases. The study was carried out in the Molecular Genetics Laboratory of the School of Biological Sciences at Universidad Nacional, Heredia, Costa Rica. The technical electrophoresis in gel of polyacrylamida SDS-PAGE was used. It was difference in the pattern of isoenzymes between both species. According to previous molecular studies based on morphology and anatomy, flavonoides, isoenzymes, and DNA sequences of certain genes both species are very different; in addition they belong to different monophyletics groups. On the other hand, the importance of this molecular characterization is its possible use in water treatment, because the esterases have had important in the bioremediation. Also, species of the Lemnaceae family are used in the water treatment like bioremediation.

Differential impact of acclimation and acute water deprivation in the expression of key transcription factors in soybean roots.

2016

Peptide-based low molecular weight gelators for the preparation of self-assembled materials

Low molecular weight gelators (LMWGs) based on pseudo-peptides are here studied for the preparation of supramolecular materials. These compounds can self-assemble through non-covalent interactions such as hydrogen bonds and π-π stacking, forming fibres and gels. A wide variety of materials can be prepared starting from these building blocks, which can be tuned and functionalised depending on the application. In this work, derivatives of the three aromatic amino acids L-Phenylalanine, L-Tyrosine and L-DOPA (3,4-dihydroxiphenylalanine) were synthesised and tested as gelators for water or organic solvents. First, the optimal gelating conditions were studied for each compound, varying concentration, solvent and trigger. Then the materials were characterised in terms of mechanical properties and morphology. Water remediation from dye pollution was the first focus of this work. Organogels were studied as absorbent of dyes from contaminated water. Hydrogels functionalised with TiO2 nanoparticles and graphene platelets were proposed as efficient materials for the photo-degradation of dyes. An efficient method for the incorporation of graphene inside hydrogels using the gelator itself as dispersant was proposed. In these materials a high storage modulus coexists with good self-healing and biocompatibility. The incorporation of a mineral phase inside the gel matrix was then investigated, leading to the preparation of composite organic/inorganic materials. In a first study, the growth of calcium carbonate crystals was achieved inside the hydrogel, which preserved its structure after crystal formation. Then the self-assembled fibres made of LMWGs were used for the first time instead of the polymeric ones as reinforcement inside calcium phosphate cements (CPCs) for bone regeneration. Gel-to-crystal transitions occurring with time in a metastable gel were also examined. The formation of organic crystals in gels can be achieved in multicomponent systems, in which a second gelator constitutes the independent gel network. Finally, some compounds unable to gelate were tested as underwater adhesives.

Molecular mechanisms controlling physiological plasticity in marine mussels under the influence of natural and anthropogenic stress factors

Marine mussels are exceptionally well-adapted to live in transitional habitats where they are exposed to fluctuating environmental parameters and elevated levels of natural and anthropogenic stressors throughout their lifecycle. However, there is a dearth of information about the molecular mechanisms that assist in dealing with environmental changes. This project aims to investigate the molecular mechanisms governing acclimatory and stress responses of the Mediterranean mussel (Mytilus galloprovincialis) by addressing relevant life stages and environmental stressors of emerging concern. The experimental approach consisted of two phases to explore (i) the physiological processes at early life history and the consequences of plastic pollution and (ii) the adult physiology processes under natural habitats. As the first phase, I employed a plastic leachate (styrene monomer), and polystyrene microplastics to understand the modulation of cytoprotective mechanisms during the early embryo stages. Results revealed the onset of transcriptional impairments of genes involved in MXR-related transporters and other physiological processes induced by styrene and PS-MPs. In the second phase, as a preliminary analysis, microbiota profile of adult mussels at the tissue scale and its surrounding water was explored to understand microbiota structures that may reflect peculiar adaptations to the respective tissue functions. The broader experiment has been implemented to understand the variability of transcriptional profiles in the mussel digestive glands in the natural setting. All the genes employed in this study have shown possibilities to use as molecular biomarker responses throughout the year for monitoring the physiology of mussels living in a particular environment and, in turn, more properly detecting changes in the environment. As a whole, my studies provide insights into the interactions between environmental parameters, and intrinsic characters, and physiology of marine bivalves, and it could help to interpretation of responses correctly under stress conditions and climate change scenarios.

Towards the conservation of vulnerable marine large predators: morphometric, molecular and microchemical variation of historical sawfish rostra from Mediterranean collections

Sawfishes (Chondrichthyes, Pristidae) are considered one of the most endangered families among elasmobranchs. Extensive efforts are required worldwide to gather solid information on historical and recent changes in the composition/range of species. In this study, we have implemented an integrative approach to characterize the species diversity and the abundance of historical rostra of sawfishes from museums and private collections of the Mediterranean area. The identification at the species level of 172 dried rostra was carried out through the integration of both traditional and geometric morphometric techniques with molecular tools, allowing the assessment of a robust methodical approach to discriminate species. In addition, we analysed 35 rostral teeth to clarify the past distribution of sawfish species considering the isotopic composition of oxygen and carbon. The morphometric, molecular, and geographical characterization of samples was accompanied by the preliminary evaluation of growth structures and the inspection of the strontium isotope composition in two teeth to unravel movement patterns of individuals across different salinities of water. Results were integrated with currently available data from public repositories and showed that the historical specimens belonged to four nominal species: Pristis zijsron (81), Anoxypristis cuspidata (39), P. pristis (30), and P. pectinata (22). An identification error of 5.41% emerged in the morphological distinction of rostra between juvenile individuals of P. pectinata and P. zijsron. The new approach of carbon and oxygen isotopes, implemented for the first time in these taxa, permitted the identification of the high-probability habitat preferences of these benthopelagic elasmobranchs in about 50% of the analysed specimens. Using this multidisciplinary approach, we successfully assigned the numerous museum rostra with lacking data to a given species and identified their candidate geographical origin, retrieving novel information and data for understanding the species distribution and ecology of past, sometimes locally/regionally extinct sawfish faunas.

Non-ambient solid-state characterization of crystalline molecular organic semiconductors

The research project is focused on the investigation of the polymorphism of crystalline molecular material for organic semiconductor applications under non-ambient conditions, and the solid-state characterization and crystal structure determination of the different polymorphic forms. In particular, this research project has tackled the investigation and characterization of the polymorphism of perylene diimides (PDIs) derivatives at high temperatures and pressures, in particular N,N’-dialkyl-3,4,9,10-perylendiimide (PDI-Cn, with n = 5, 6, 7, 8). These molecules are characterized by excellent chemical, thermal, and photostability, high electron affinity, strong absorption in the visible region, low LUMO energies, good air stability, and good charge transport properties, which can be tuned via functionalization; these features make them promising n-type organic semiconductor materials for several applications such as OFETs, OPV cells, laser dye, sensors, bioimaging, etc. The thermal characterization of PDI-Cn was carried out by a combination of differential scanning calorimetry, variable temperature X-ray diffraction, hot-stage microscopy, and in the case of PDI-C5 also variable temperature Raman spectroscopy. Whereas crystal structure determination was carried out by both Single Crystal and Powder X-ray diffraction. Moreover, high-pressure polymorphism via pressure-dependent UV-Vis absorption spectroscopy and high-pressure Single Crystal X-ray diffraction was carried out in this project. A data-driven approach based on a combination of self-organizing maps (SOM) and principal component analysis (PCA) is also reported was used to classify different π-stacking arrangements of PDI derivatives into families of similar crystal packing. Besides the main project, in the framework of structure-property analysis under non-ambient conditions, the structural investigation of the water loss in Pt- and Pd- based vapochromic potassium/lithium salts upon temperature, and the investigation of structure-mechanical property relationships in polymorphs of a thienopyrrolyldione endcapped oligothiophene (C4-NT3N) are reported.

A glassful of graphene: graphene-based materials for drinking water remediation

Modern world suffers from an intense water crisis. Emerging contaminants represent one of the most concerning elements of this issue. Substances, molecules, ions, and microorganisms take part in this vast and variegated class of pollutants, which main characteristic is to be highly resistant to traditional water purification technologies. An intense international research effort is being carried out in order to find new and innovative solutions to this problem, and graphene-based materials are one of the most promising options. Graphene oxide (GO) is a nanostructured material where domains populated by oxygenated groups alternate with interconnected areas of sp2 hybridized carbon atoms, on the surface of a one-atom thick nanosheets. GO can adsorb a great number of molecules and ions on its surface, thanks to the variety of different interactions that it can express, such as hydrogen bonding, p-p stacking, and electrostatic and hydrophobic interaction. These characteristics, added to the high superficial area, make it an optimal material for the development of innovative materials for drinking water remediation. The main concern in the use of GO in this field is to avoid secondary contaminations (i.e. GO itself must not become a pollutant). This issue can be faced through the immobilization of GO onto polymeric substrates, thus developing composite materials. The use of micro/ultrafiltration polymeric hollow fibers as substrates allows the design of adsorptive membranes, meaning devices that can perform filtration and adsorption simultaneously. In this thesis, two strategies for the development of adsorptive membranes were investigated: a core-shell strategy, where hollow fibers are coated with GO, and a coextrusion strategy, where GO is embedded in the polymeric matrix of the fibers. The so-obtained devices were exploited for both fundamental studies (i.e. molecular and ionic behaviour in between GO nanosheets) and real applications (the coextruded material is now at TRL 9).

Investigation of organic semiconductor/water interfaces for optobioelectronic devices

The thesis investigates the potential of photoactive organic semiconductors as a new class of materials for developing bioelectronic devices that can convert light into biological signals. The materials can be either small molecules or polymers. When these materials interact with aqueous biological fluids, they give rise to various electrochemical phenomena, including photofaradaic or photocapacitive processes, depending on whether photogenerated charges participate in redox processes or accumulate at an interface. The thesis starts by studying the behavior of the H2Pc/PTCDI molecular p/n thin-film heterojunction in contact with aqueous electrolyte. An equivalent circuit model is developed, explaining the measurements and predicting behavior in wireless mode. A systematic study on p-type polymeric thin-films is presented, comparing rr-P3HT with two low bandgap conjugated polymers: PBDB-T and PTB7. The results demonstrate that PTB7 has superior photocurrent performance due to more effective electron-transfer onto acceptor states in solution. Furthermore, the thesis addresses the issue of photovoltage generation for wireless photoelectrodes. An analytical model based on photoactivated charge-transfer across the organic-semiconductor/water interface is developed, explaining the large photovoltages observed for polymeric p-type semiconductor electrodes in water. Then, flash-precipitated nanoparticles made of the same three photoactive polymers are investigated, assessing the influence of fabrication parameters on the stability, structure, and energetics of the nanoparticles. Photocathodic current generation and consequent positive charge accumulation is also investigated. Additionally, newly developed porous P3HT thin-films are tested, showing that porosity increases both the photocurrent and the semiconductor/water interfacial capacity. Finally, the thesis demonstrates the biocompatibility of the materials in in-vitro experiments and shows safe levels of photoinduced intracellular ROS production with p-type polymeric thin-films and nanoparticles. The findings highlight the potential of photoactive organic semiconductors in the development of optobioelectronic devices, demonstrating their ability to convert light into biological signals and interface with biological fluids.

Diazene photoswitches in artificial molecular machines and light-effected materials

The research developed in this thesis focused on the spectroscopic and photochemical characterization of molecular diazene photoswitches, both as individual species and as functional components of mechanically interlocked molecules, molecular-based materials and artificial molecular machines and motors. Among the plethora of photochromes reported so far, azobenzene is the most versatile photoswitch due to its reproducible and well-established photochemical properties. Part I of this thesis work focuses on the characterization of light-responsive supramolecular systems based on azobenzene: a photochemically-driven rotary motor, a light-responsive supramolecular polymeric material and a supramolecular system capable of photoinduced entantiodiscrimination. Despite the wide success of azobenzene photoswitches, the tunability of their photochemical properties as a function of the diversified substitution pattern on its aryl ring presents intrinsic limitations. To overcome this issue, in the last decade heteroaryl azoswitches (i.e., azobenzene having heterocyclic rings in place of one or both phenyl groups) have attracted a great deal of attention. Hence, Part II of this thesis work treats the photochemical characterization of two different families of azoheteroarenes embedding imidazolium and thienyl functionalities in their structures. Their potential implementation in water-soluble artificial molecular machines and light-effected semiconductor materials is also assessed.

Low-molecular-weight gels: from the rational design to the application of versatile soft materials

Low-molecular-weight (LMW) gels are a versatile class of soft materials that gained increasing interest over the last few decades. They are made of a small percentage, often lower than 1.0 %, of organic molecules called gelators, dispersed in a liquid medium. Such molecules have a molecular weight usually lower than 1 kDa. The gelator molecules start to interact after the addition of a trigger, and form fibres, whose entanglement traps the solvent through capillary forces. A plethora of LMW gelators have been designed, including short peptides. Such gelators present several advantages: the synthesis is easy and can be easily scaled up; they are usually biocompatible and biodegradable; the gelation phenomenon can be rationalised by making small variation on the peptide scaffold; they find application in several fields. In this thesis, an overview of several peptide based LMW gels is presented. In each study, the gelation conditions were carefully studied, and the final materials were thoroughly investigated. First, the gelation ability of a fluorinated phenylalanine was assessed, to understand how the presence of a rigid moiety and the presence of fluorine may influence the gelation. In this context, a method for the dissolution of sensitive gelators was studied. Then, the control over the gel formation was studied both over time and space, taking advantage of either the pH-annealing of the gel or the reaction-diffusion of a hydrolysing reagent. Some gels were probed for various applications. Due to their ability of trapping water and organic solvents, we used gels for trapping pollutants dissolved in water, as well as a medium for the controlled release of either fragrances or bioactive compounds. Finally, the interaction of the gel matrix with a light-responsive molecule was assessed to understand wether the gel properties or the interaction of the additive with light were affected.

Exploiting molecular simulations as an atomic-level microscope to elucidate clinically relevant cation chloride cotransporters

The cation chloride cotransporters (CCCs) represent a vital family of ion transporters, with several members implicated in significant neurological disorders. Specifically, conditions such as cerebrospinal fluid accumulation, epilepsy, Down’s syndrome, Asperger’s syndrome, and certain cancers have been attributed to various CCCs. This thesis delves into these pharmacological targets using advanced computational methodologies. I primarily employed GPU-accelerated all-atom molecular dynamics simulations, deep learning-based collective variables, enhanced sampling methods, and custom Python scripts for comprehensive simulation analyses. Our research predominantly centered on KCC1 and NKCC1 transporters. For KCC1, I examined its equilibrium dynamics in the presence/absence of an inhibitor and assessed the functional implications of different ion loading states. In contrast, our work on NKCC1 revealed its unique alternating access mechanism, termed the rocking-bundle mechanism. I identified a previously unobserved occluded state and demonstrated the transporter's potential for water permeability under specific conditions. Furthermore, I confirmed the actual water flow through its permeable states. In essence, this thesis leverages cutting-edge computational techniques to deepen our understanding of the CCCs, a family of ion transporters with profound clinical significance.

Thermodynamic Study Of The Micellization Of Zwitterionic Surfactants And Their Interaction With Polymers In Water By Isothermal Titration Calorimetry.

The micellization of a homologous series of zwitterionic surfactants, a group of sulfobetaines, was studied using isothermal titration calorimetry (ITC) in the temperature range from 15 to 65 °C. The increase in both temperature and the alkyl chain length leads to more negative values of ΔGmic(0) , favoring the micellization. The entropic term (ΔSmic(0)) is predominant at lower temperatures, and above ca. 55-65 °C, the enthalpic term (ΔHmic(0)) becomes prevalent, figuring a jointly driven process as the temperature increases. The interaction of these sulfobetaines with different polymers was also studied by ITC. Among the polymers studied, only two induced the formation of micellar aggregates at lower surfactant concentration: poly(acrylic acid), PAA, probably due to the formation of hydrogen bonds between the carboxylic group of the polymer and the sulfonate group of the surfactant, and poly(sodium 4-styrenesulfonate), PSS, probably due to the incorporation of the hydrophobic styrene group into the micelles. The prevalence of the hydrophobic and not the electrostatic contributions to the interaction between sulfobetaine and PSS was confirmed by an increased interaction enthalpy in the presence of electrolytes (NaCl) and by the observation of a significant temperature dependence, the latter consistent with the proposed removal of hydrophobic groups from water.

Molecular And Biochemical Characterization Of Caffeine Synthase And Purine Alkaloid Concentration In Guarana Fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.