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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.

Convolutional Neural Network Architectures for Template Matching

The Neural Networks customized and tested in this thesis (WaldoNet, FlowNet and PatchNet) are a first exploration and approach to the Template Matching task. The possibilities of extension are therefore many and some are proposed below. During my thesis, I have analyzed the functioning of the classical algorithms and adapted with deep learning algorithms. The features extracted from both the template and the query images resemble the keypoints of the SIFT algorithm. Then, instead of similarity function or keypoints matching, WaldoNet and PatchNet use the convolutional layer to compare the features, while FlowNet uses the correlational layer. In addition, I have identified the major challenges of the Template Matching task (affine/non-affine transformations, intensity changes...) and solved them with a careful design of the dataset.

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.

Development And Characterization Of A Cationic Lipid Nanocarrier As Non-viral Vector For Gene Therapy.

The aim of the present work was to produce a cationic solid lipid nanoparticle (SLN) as non-viral vector for protein delivery. Cationic SLN were produced by double emulsion method, composed of softisan(®) 100, cetyltrimethylammonium bromide (CTAB), Tween(®) 80, Span(®) 80, glycerol and lipoid(®) S75 loading insulin as model protein. The formulation was characterized in terms of mean hydrodynamic diameter (z-ave), polydispersity index (PI), zeta potential (ZP), stability during storage time, stability after lyophilization, effect of toxicity and transfection ability in HeLa cells, in vitro release profile and morphology. SLN were stable for 30days and showed minimal changes in their physicochemical properties after lyophilization. The particles exhibited a relatively slow release, spherical morphology and were able to transfect HeLa cells, but toxicity remained an obstacle. Results suggest that SLN are nevertheless promising for delivery of proteins or nucleic acids for gene therapy.

Molecularly Imprinted Solid Phase Extraction Of Fluconazole From Pharmaceutical Formulations.

This work encompasses a direct and coherent strategy to synthesise a molecularly imprinted polymer (MIP) capable of extracting fluconazole from its sample. The MIP was successfully prepared from methacrylic acid (functional monomer), ethyleneglycoldimethacrylate (crosslinker) and acetonitrile (porogenic solvent) in the presence of fluconazole as the template molecule through a non-covalent approach. The non-imprinted polymer (NIP) was prepared following the same synthetic scheme, but in the absence of the template. The data obtained from scanning electronic microscopy, infrared spectroscopy, thermogravimetric and nitrogen Brunauer-Emmett-Teller plot helped to elucidate the structural as well as the morphological characteristics of the MIP and NIP. The application of MIP as a sorbent was demonstrated by packing it in solid phase extraction cartridges to extract fluconazole from commercial capsule samples through an offline analytical procedure. The quantification of fluconazole was accomplished through UPLC-MS, which resulted in LOD≤1.63×10(-10) mM. Furthermore, a high percentage recovery of 91±10% (n=9) was obtained. The ability of the MIP for selective recognition of fluconazole was evaluated by comparison with the structural analogues, miconazole, tioconazole and secnidazole, resulting in percentage recoveries of 51, 35 and 32%, respectively.

Properties Of Bologna-type Sausages With Pork Back-fat Replaced With Pork Skin And Amorphous Cellulose.

Bologna-type sausages were produced with 50% of their pork back-fat content replaced with gels elaborated with different ratios of pork skin, water, and amorphous cellulose (1:1:0, 1:1:0.1, 1:1:0.2, 1:1:0.3, and 1:1:0.4). The impact of such replacement on the physico-chemical characteristics and the consumer sensory profiling was evaluated. The modified treatments had 42% less fat, 18% more protein, and 8% more moisture than the control group. Treatments with amorphous cellulose had a lower cooking loss and higher emulsion stability. High amorphous cellulose content (1:1:0.3 and 1:1:0.4) increased hardness, gumminess, and chewiness. The gel formulated with the ratio of 1:1:0.2 (pork skin: water: amorphous cellulose gel) provided a sensory sensation similar to that provided by fat and allowed products of good acceptance to be obtained. Therefore, a combination of pork skin and amorphous cellulose is useful in improving technological quality and producing healthier and sensory acceptable bologna-type sausages.

Hemodynamic Changes With Two Lipid Emulsions For Treatment Of Bupivacaine Toxicity In Swines.

To compare the hemodynamic changes following two different lipid emulsion therapies after bupivacaine intoxication in swines. Large White pigs were anesthetized with thiopental, tracheal intubation performed and mechanical ventilation instituted. Hemodynamic variables were recorded with invasive pressure monitoring and pulmonary artery catheterization (Swan-Ganz catheter). After a 30-minute resting period, 5 mg.kg-1 of bupivacaine by intravenous injection was administered and new hemodynamic measures were performed 1 minute later; the animals were than randomly divided into three groups and received 4 ml.kg-1 of one of the two different lipid emulsion with standard long-chaim triglyceride, or mixture of long and medium-chain triglyceride, or saline solution. Hemodynamic changes were then re-evaluated at 5, 10, 15, 20 and 30 minutes. Bupivacaine intoxication caused fall in arterial blood pressure, cardiac index, ventricular systolic work index mainly and no important changes in vascular resistances. Both emulsion improved arterial blood pressure mainly increasing vascular resistance since the cardiac index had no significant improvement. On the systemic circulation the hemodynamic results were similar with both lipid emulsions. Both lipid emulsions were efficient and similar options to reverse hypotension in cases of bupivacaine toxicity.

Breaking Oil-in-water Emulsions Stabilized By Yeast.

Several biotechnological processes can show an undesirable formation of emulsions making difficult phase separation and product recovery. The breakup of oil-in-water emulsions stabilized by yeast was studied using different physical and chemical methods. These emulsions were composed by deionized water, hexadecane and commercial yeast (Saccharomyces cerevisiae). The stability of the emulsions was evaluated varying the yeast concentration from 7.47 to 22.11% (w/w) and the phases obtained after gravity separation were evaluated on chemical composition, droplet size distribution, rheological behavior and optical microscopy. The cream phase showed kinetic stability attributed to mechanisms as electrostatic repulsion between the droplets, a possible Pickering-type stabilization and the viscoelastic properties of the concentrated emulsion. Oil recovery from cream phase was performed using gravity separation, centrifugation, heating and addition of demulsifier agents (alcohols and magnetic nanoparticles). Long centrifugation time and high centrifugal forces (2h/150,000×g) were necessary to obtain a complete oil recovery. The heat treatment (60°C) was not enough to promote a satisfactory oil separation. Addition of alcohols followed by centrifugation enhanced oil recovery: butanol addition allowed almost complete phase separation of the emulsion while ethanol addition resulted in 84% of oil recovery. Implementation of this method, however, would require additional steps for solvent separation. Addition of charged magnetic nanoparticles was effective by interacting electrostatically with the interface, resulting in emulsion destabilization under a magnetic field. This method reached almost 96% of oil recovery and it was potentially advantageous since no additional steps might be necessary for further purifying the recovered oil.

Produção de micropartículas de alginato contendo Flavobacterium columnare inativada pelo método de emulsão para vacinação de peixes por via oral

Alginate microparticles were prepared by an emulsion method aiming oral controlled release of antigens to fish. The effects of emulsification temperature and impeller type on particle morphology, average diameter, and size distribution were evaluated. Microparticles contaning formalin-killed Flavobacterium columnare cells (a model antigen) were prepared and characterized regarding bacterial release and particle stability when exposed to Nile tilapia (Oreochromis niloticus) typical gastrointestinal conditions. This methodology allowed the production of microparticles containing up to 14.3 g/L of bacterin, stable at a pH range from 2.0 to 9.0 for 12 h and smaller than 35 μm.

Qualidade pós-colheita de caqui 'fuyu' com utilização de diferentes concentrações de cobertura comestível

One of the main objectives of applying edible coatings on fruits surface is to create a protective film to reduce weight loss due to evaporation and transpiration and also to decrease the risk of fruit rot caused by environmental contamination, in order to improve the visual aspect. Therefore, it is possible to increase shelf life, and decrease post harvest losses. Persimmon is a much appreciated fruit, with high potential for export, but sensitive to handling and storage. This study aimed to evaluate the effect of applying the edible coating Megh Wax ECF-124 (18% of active composts, consisting of emulsion of carnauba wax, anionic surfactant, preservative and water) produced by Megh Industry and Commerce Ltda in three different concentrations (25, 50 and 100%) on post harvest quality of 'Fuyu' persimmon stored for 14 days. The attributes evaluated for quality were: firmness, pH, acidity, soluble solids, weight loss and color. The results showed that application of carnauba wax in different concentrations was effective on decreasing weight loss of persimmon cv. Fuyu and maintenance of color aspects. Treatment at lower concentration, 25%, showed lower rate of discharge, but high concentrations showed lower values of mass loss. Carnauba wax application showed a high potential for use on postharvest conservation, and can be applied together with other technologies, helping to maintain quality for export.

Characterization of thiol-functionalised silica films deposited on electrode surfaces

Thiol-functionalised silica films were deposited on various electrode surfaces (gold, platinum, glassy carbon) by spin-coating sol-gel mixtures in the presence of a surfactant template. Film formation occurred by evaporation induced self-assembly (EISA) involving the hydrolysis and (co)condensation of silane and organosilane precursors on the electrode surface. The characterization of such material was performed by IR spectroscopy, thermogravimetry (TG), elemental analysis (EA), atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV).

Produção de substratos sers eficientes através da deposição de ouro sobre um molde de microesferas de poliestireno

This work reports on the SERS activity of a nanostructured substrate that was obtained by electrodepositing gold over a template consisting of polystyrene microspheres. This substrate displayed superior SERS performance for the detection of 4-merctaptopyridine as compared to a conventional roughened Au electrode. In order to investigate the substrate capability for the detection at low concentration limits, a series of Rhodamine 6G (1 nM) spectra were registered. Our spectral dynamics data is in agreement with single-molecule behavior, showing that the control over the substrate morphology is crucial to enable the production of highly reproducible and sensitive SERS substrates.