REGULATION OF SHAPE DYNAMICS AND ACTIN POLYMERIZATION DURING COLLECTIVE CELL MIGRATION

This thesis aims to understand how cells coordinate their motion during collective migration. As previously shown, the motion of individually migrating cells is governed by wave-like cell shape dynamics. The mechanisms that regulate these dynamic behaviors in response to extracellular environment remain largely unclear. I applied shape dynamics analysis to Dictyostelium cells migrating in pairs and in multicellular streams and found that wave-like membrane protrusions are highly coupled between touching cells. I further characterized cell motion by using principle component analysis (PCA) to decompose complex cell shape changes into a serial shape change modes, from which I found that streaming cells exhibit localized anterior protrusion, termed front narrowing, to facilitate cell-cell coupling. I next explored cytoskeleton-based mechanisms of cell-cell coupling by measuring the dynamics of actin polymerization. Actin polymerization waves observed in individual cells were significantly suppressed in multicellular streams. Streaming cells exclusively produced F-actin at cell-cell contact regions, especially at cell fronts. I demonstrated that such restricted actin polymerization is associated with cell-cell coupling, as reducing actin polymerization with Latrunculin A leads to the assembly of F-actin at the side of streams, the decrease of front narrowing, and the decoupling of protrusion waves. My studies also suggest that collective migration is guided by cell-surface interactions. I examined the aggregation of Dictyostelim cells under distinct conditions and found that both chemical compositions of surfaces and surface-adhesion defects in cells result in altered collective migration patterns. I also investigated the shape dynamics of cells suspended on PEG-coated surfaces, which showed that coupling of protrusion waves disappears on touching suspended cells. These observations indicate that collective migration requires a balance between cell-cell and cell-surface adhesions. I hypothesized such a balance is reached via the regulation of cytoskeleton. Indeed, I found cells actively regulate cytoskeleton to retain optimal cell-surface adhesions on varying surfaces, and cells lacking the link between actin and surfaces (talin A) could not retain the optimal adhesions. On the other hand, suspended cells exhibited enhanced actin filament assembly on the periphery of cell groups instead of in cell-cell contact regions, which facilitates their aggregation in a clumping fashion.

UHMWPE/SBA-15 nanocomposites synthesized by in situ polymerization

Different nanocomposites have been attained by in situ polymerization based on ultra-high molecular weight polyethylene (UHMWPE) and mesoporous SBA-15, this silica being used for immobilization of the FI catalyst bis [N-(3-tert-butylsalicylidene)-2,3,4,5,6-pentafluoroanilinato] titanium (IV) dichloride and as filler as well. Two distinct approaches have been selected for supporting the FI catalyst on the SBA-15 prior polymerization. A study on polymerization activity of this catalyst has been performed under homogenous conditions and upon heterogenization. A study of the effect of presence of mesoporous particles and of the immobilization method is also carried out. Moreover, the thermal characterization, phase transitions and mechanical response of some pristine UHMWPEs and UHMWPE/SBA-15 materials have been carried out. Relationships with variations on molar mass, impregnation method of catalyst and final SBA-15 content have been established.

Hybrid materials based on polyethylene and MCM-41 microparticles functionalized with silanes: catalytic aspects of in situ polymerization, crystalline features and mechanical properties

New nanocomposites based on polyethylene have been prepared by in situ polymerization of ethylene in presence of mesoporous MCM-41. The polymerization reactions were performed using a zirconocene catalyst either under homogenous conditions or supported onto mesoporous MCM-41 particles, which are synthesized and decorated post-synthesis with two silanes before polymerization in order to promote an enhanced interfacial adhesion. The existence of polyethylene chains able to crystallize within the mesoporous channels in the resulting nanocomposites is figured out from the small endothermic process, located at around 80 C, on heating calorimetric experiments, in addition to the main melting endotherm. These results indicate that polyethylene macrochains can grow up during polymerization either outside or inside the MCM-41 channels, these keeping their regular hexagonal arrangements. Mechanical response is observed to be dependent on the content in mesoporous MCM-41 and on the crystalline features of polyethylene. Accordingly, stiffness increases and deformability decreases in the nanocomposites as much as MCM-41 content is enlarged and polyethylene amount within channels is raised. Ultimate mechanical performance improves with MCM-41 incorporation without varying the final processing temperature.

Ziziphus mauritiana leaf extract emulsion for skin rejuvenation

Purpose: To formulate stable water in oil (W/O) emulsion containing hydroalcoholic crude extract of Ziziphus mauritiana leaves for skin rejuvenation. Methods: Placebo (base) without any plant extract and formulation with 4 % Ziziphus mauritiana extract were prepared by mixing. Samples of the emulsions were subjected to varying storage conditions, i.e., 8, 25, 40 oC and 40 oC + 75 % relative humidity for a period of 4 weeks to predict their stability. During this period, stability parameters, including liquefaction, phase separation, color, electrical conductivity, centrifugation and pH were monitored at specified time intervals. Skin rejuvenation was evaluated using 13 healthy human volunteers over a period of 8 weeks. During this period, various skin parameters such as erythema, melanin level, moisture content, elasticity and sebum content of the skin were evaluated at specified intervals. Results: Both the active formulation and placebo were stable in terms of liquifaction, phase separation and color at all the storage conditions of temperature and humidity. Active formulation showed statistically significant (p < 0.05) improvement in skin melanin as well as in skin moisture and sebum levels, whereas these properties were reduced or even absent in the placebo formulation (p > 0.05). Both active and placebo formulations changed skin elasticity and erythema significantly (p < 0.05). Conclusion: It is evident from the findings that the leaf extract of Ziziphus mauritiana possesses antiaging properties as well as exert skin lightening, moisturizing and viscoelastic effects on human skin.

Formulation and stability of topical water in oil emulsion containing corn silk extract

Purpose: To formulate the water in oil (W/O) emulsion of corn silk (CS) extract and to evaluate its stability at various storage conditions. Methods: Ethanol CS extract was prepared using maceration (cold) technique. A 4 % CS emulsion was prepared using varying concentrations of liquid paraffin, ABIL EM90 and water. The formulations were kept at 40 oC for 28 days and to screen out the less stable formulations. The remaining formulations were further stressed at 50 oC to choose the most stable formulation. The optimized formulation was evaluated for physical characteristics including phase separation, rheology and mean droplet size. The physical stability of the formulation was evaluated by monitoring these parameters over a period of 12 weeks at 8, 25, 40 and 40 oC, and 75 % RH. Results: The chosen formulation showed good resistance to phase separation on centrifugation under all storage conditions. Rheological behavior followed non-Newtonian pseudoplastic pattern at various storage conditions. Mean droplet size of freshly prepared formulation was 2.98 ± 1.32 µm and did not show significant (p < 0.05) changes at normal storage conditions (8 and 25 oC). Conclusion: The findings indicate that the developed CS extract W/O emulsion is stable and therefore may be suitable for topical use on skin as an antioxidant preparation.

A new post-metallocene-ti catalyst with maltolate bidentade ligand: an investigation in heterogeneous polymerization reactions in different mesoporous supports

A new titanium catalyst easily synthesized from ethylmaltol bidentate chelator ligand was studied in homogeneous and heterogeneous ethylene polymerization. The dichlorobis(3-hydroxy-2-ethyl-4-pyrone)titanium(IV) complex was characterized by 1H and 13C NMR (nuclear magnetic resonance), UV-Vis and elemental analysis. Theoretical study by density functional theory (DFT) showed that the complex chlorines exhibit cis configuration, which is important for the activity in olefin polymerization. The complex was supported by two methods, direct impregnation or methylaluminoxane (MAO) pre-treatment, in five mesoporous supports: MCM-41 (micro and nano), SBA-15 and also the corresponding modified Al species. All the catalytic systems were active in ethylene polymerization and the catalytic activity was strongly influenced by the method of immobilization of the catalyst and the type of support.

Hafnocene catalyst for polyethylene and its nanocomposites with SBA-15 by in situ polymerization: immobilization approaches, catalytic behavior and properties evaluation

A hafnocene catalyst combined with methylaluminoxane (MAO) has been used as catalytic complex for the preparation of a set of polyethylene homopolymers by in situ polymerization under homogenous conditions and of different nanocomposites with mesoporous SBA- 15 particles, the latter playing the dual role of catalyst support and nanofiller. Distinct immobilization approaches have been explored for obtainment of these nanocomposites. Moreover, catalytic features, thermal stability, melting and crystallization transitions and mechanical behavior have been evaluated for those materials.

Adenine as an organocatalyst for the ring-opening polymerization of lactide: scope, mechanism and access to adenine-functionalized polylactide

Nucleobase-functionalized polymers are widely used in the fields of supramolecular chemistry and self-assembly, and their development for biomedical applications is also an area of interest. They are usually synthesized by tedious multistep procedures. In this study, we assess adenine as an organoinitiator/ organocatalyst for the ring-opening polymerization of lactide. L-Lactide can be quantitatively polymerized in the presence of adenine. Reaction conditions involving short reaction times and relatively low temperatures enable the access to adenine end-capped polylactide in a simple one-step procedure, in bulk, without additional catalyst. DFT calculations show that the polymerization occurs via hydrogen bond catalysis. The mechanism involves (i) a hydrogen bond between the NH9 of adenine and the carbonyl moiety of lactide, leading to an electron deficient carbon atom, and (ii) a second hydrogen bond between the N3 of adenine and the NH2 of a second adenine molecule, followed by a nucleophilic attack of the latter activated amine on the former electron deficient carbon on the monomer. For longer reaction times and higher temperatures, macrocyclic species are formed, and a mechanism involving the imidazole ring of adenine is proposed based on literature studies. Depending on the reaction conditions, adenine can thus be considered as an organoinitiator or an organocatalyst for the ring-opening polymerization of lactide.

Adenine as an organocatalyst for the ring-opening polymerization of lactide: scope, mechanism and access to adenine-functionalized polylactide

Nucleobase-functionalized polymers are widely used in the fields of supramolecular chemistry and self-assembly, and their development for biomedical applications is also an area of interest. They are usually synthesized by tedious multistep procedures. In this study, we assess adenine as an organoinitiator/organocatalyst for the ring-opening polymerization of lactide. L-Lactide can be quantitatively polymerized in the presence of adenine. Reaction conditions involving short reaction times and relatively low temperatures enable the access to adenine end-capped polylactide in a simple one-step procedure, in bulk, without additional catalyst. DFT calculations show that the polymerization occurs via hydrogen bond catalysis. The mechanism involves (i) a hydrogen bond between the NH9 of adenine and the carbonyl moiety of lactide, leading to an electron deficient carbon atom, and (ii) a second hydrogen bond between the N3 of adenine and the NH2 of a second adenine molecule, followed by a nucleophilic attack of the latter activated amine on the former electron deficient carbon on the monomer. For longer reaction times and higher temperatures, macrocyclic species are formed, and a mechanism involving the imidazole ring of adenine is proposed based on literature studies. Depending on the reaction conditions, adenine can thus be considered as an organoinitiator or an organocatalyst for the ring-opening polymerization of lactide.

DFT STUDIES ON THE MECHANISM OF RING-OPENING POLYMERIZATION OF LACTIDE WITH ADENINE

The use of organic molecules as catalysts for the ring-opening polymerization (ROP) of cyclic esters has gained much interest last years.[1] The use of a molecule of biological interest, able to initiate ROP of cyclic esters without any cocatalyst is even more interesting, as the resulting material will not contain any catalytic residue. Nucleobase-polymer conjugates development is thus an emerging area envisaging biomedical applications.[2] However, they are usually synthesized by tedious multistep procedures. Recently, adenine was used as organoinitiator for the ROP of L-lactide.[3] Reaction conditions involving short reaction times and relatively low temperatures enable the access to adenine-polylactide(Adn-PLA)conjugates in a simple one-step procedure, without additional catalyst and in the absence of solvent. In this study, computational investigations with density functional theory (DFT) were performed in order to clarify the reaction mechanism leading to the desired Adn-PLA. The results show that a hydrogen bond catalytic mechanism, involving a nucleophilic attack of the activated amine group of adenine onto the carbonyl group of lactide, seem to be plausible.

Design, development and characterization of DHA-based emulsion for Nutrilipidomics strategies

The industrial PhD project presented here is part of the R&D strategies of the Lipinutragen company. The innovation brought by the company concerns nutrilipidomics, i.e. the correlation between the lipid composition (in fatty acids) of the cell membrane and lipid-based nutraceuticals, especially starting from the well-known dependence of the lipid composition on the intake of essential fats, omega- 6 and omega-3 polyunsaturated fatty acids. Among the results obtained from the membrane lipidomic profiles, the case of autistic subjects is here highlighted, showing the significant deficiency of docosahexaenoic acid (DHA). The activity during the PhD was devoted to the nutrilipidomic approach. Part of the activities were devoted to scientific research in lipidomics: a) the study of lipidomic profiles in the frame of two collaboration projects: one with the group of Dr. I. Tueros at AZTI, Bilbao, regading obese population, and the other one regarding seed germination with the changes of the fatty acid profiles with the group of prof. A. Balestrazzi of the University of Parma; b) the liposome preparation for protection and lifetime prolongation of the peptide somatostatin, which was an important premise to the formulation of the DHA-containing microemulsion. The activities was also focused on the development of DHA-containing nutraceutical formulations in the form of emulsion, overcoming the difficulty of the capsule ingestion, to be administered orally. The work pointed to study the combination of active ingredients, based on the previous know-how regarding the bioavailability for the cell membrane incorporation. The ingredients of the formulation were studied and tested in vitro for the bioavailability of DHA to be incorporated in the cell membranes of different types of cultured cells. Part of this study is covered by non-disclosure agreement since it belongs to the know-how of Lipinutragen.

Study and implementation of control strategies for polymerization processes assisted by atmospheric pressure plasma jets

In recent years, polymerization processes assisted by atmospheric pressure plasma jets (APPJs) have received increasing attention in numerous industrially relevant sectors since they allow to coat complex 3D substrates without requiring expensive vacuum systems. Therefore, advancing the comprehension of these processes has become a high priority topic of research. This PhD dissertation is focused on the study and the implementation of control strategies for a polymerization process assisted by an atmospheric pressure single electrode plasma jet. In the first section, a study of the validity of the Yasuda parameter (W/FM) as controlling parameter in the polymerization process assisted by the plasma jet and an aerosolized fluorinated silane precursor is proposed. The surface characterization of coatings deposited under different W/FM values reveals the presence of two very well-known deposition domains, thus suggesting the validity of W/FM as controlling parameter. In addition, the key role of the Yasuda parameter in the process is further demonstrated since coatings deposited under the same W/FM exhibit similar properties, regardless of how W/FM is obtained. In the second section, the development of a methodology for measuring the energy of reactions in the polymerization process assisted by the plasma jet and vaporized hexamethyldisiloxane is presented. The values of energy per precursor molecule are calculated through the identification and resolution of a proper equivalent electrical circuit. To validate the methodology, these energy values are correlated to the bond energies in the precursor molecule and to the properties of deposited thin films. It is shown that the precursor fragmentation in the discharge and the coating characteristics can be successfully explained according to the obtained values of energy per molecule. Through a detailed discussion of the limits and the potentialities of both the control strategies, this dissertation provides useful insights into the control of polymerization processes assisted by APPJs.

Polymerization of isatin towards polymers for anion exchange membranes and gas separation applications

In a world where the problem of energy resources, pollution and all aspects related to these issues become more and more dominant, a greater commitment is needed in the search for solutions. The goal of this project is to make a contribution to the research and development of new materials to reduce the environmental impact in some fields. First of all, we tried to synthesize and prepare an isatin-based membrane which has the potential for use in separating industrial gases. Furthermore, ion exchange membranes, specifically hydroxide exchange membranes (HEMs) derived from the same product can be developed for fuel cells (HEMFC) applications. These materials are essential for energy conversion and storage. The most difficult challenge is to guarantee their thermal stability and stability in corrosive environments such as alkali without losing efficiency. In recent years the poly- hydroxyalkylation catalysed with superacids, e.g. TFSA, has become increasingly studied. This reaction is exploited for the synthesis of the compounds of this thesis. After a preliminary optimization of the reaction conditions it was concluded that due to the rigidity and excessive reactivity of the system, it was not possible to obtain the isatin-based membrane to evaluate the gas separation properties. The synthesis of precursor materials for HEMs was successful by using 1-(4-bromobutyl)indoline-2,3-dione (BID) instead of isatin. A characterization of the obtained polymers was carried out using NMR, TGA and DSC analyses, and subsequently the membranes were functionalized with different ammonium-based cations. Unfortunately, this last step was not successful due to the appearance of side reactions. Future studies on the mechanism and kinetics of the reaction solve this obstacle.

Solid Lipid Nanoparticles For Hydrophilic Biotech Drugs: Optimization And Cell Viability Studies (caco-2 & Hepg-2 Cell Lines).

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.