Production of secondary metabolites in cell suspension cultures of Ocimum sanctum L.

Dissertação de mestrado em Plant Molecular Biology, Biotechnology and Bioentrepreneurship

Bioactivity of phenolic acids: Metabolites versus parent compounds: A review

Phenolic acids are present in our diet in different foods. In particular, mushrooms are a good source of these molecules. Due to their bioactive properties, phenolic acids are extensively studied and there is evidence of their role in disease prevention. Nevertheless, in vivo, these compounds are metabolized and circulate in the organism as glucuronated, sulfated and methylated metabolites, displaying higher or lower bioactivity. To clarify the importance of the metabolism of phenolic acids, the knowledge about the bioactivity of the metabolites is extremely important. In this review, chemical features, biosynthesis and bioavailability of phenolic acids are discussed as well as the chemical and enzymatic synthesis of their metabolites. Finally, the metabolites bioactive properties are compared with that of the corresponding parental compounds.

Synthesis, antiangiogenesis evaluation and molecular docking studies of 1-Aryl-3-[(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas: Discovery of a new substitution pattern for type II VEGFR-2 Tyr kinase inhibitors

The synthesis and biological evaluation of novel 1-aryl-3-[2-, 3- or 4-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 3, 4 and 5 as VEGFR-2 tyrosine kinase inhibitors, are reported. The 1-aryl-3-[3-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 4a-4h, with the arylurea in the meta position to the thioether, showed the lowest IC50 values in enzymatic assays (10-206 nM), the most potent compounds 4d-4h (IC50 10-28 nM) bearing hydrophobic groups (Me, F, CF3 and Cl) in the terminal phenyl ring. A convincing rationalization was achieved for the highest potent compounds 4 as type II VEGFR-2 inhibitors, based on the simultaneous presence of: (1) the thioether linker and (2) the arylurea moiety in the meta position. For compounds 4, significant inhibition of Human Umbilical Vein Endothelial Cells (HUVECs) proliferation (BrdU assay), migration (wound-healing assay) and tube formation were observed at low concentrations. These compounds have also shown to increase apoptosis using the TUNEL assay. Immunostaining for total and phosphorylated (active) VEGFR-2 was performed by Western blotting. The phosphorylation of the receptor was significantly inhibited at 1.0 and 2.5 microM for the most promising compounds. Altogether, these findings point to an antiangiogenic effect in HUVECs.

Laccase-catalyzed synthesis of conducting polyaniline-lignosulfonate composite

Enzymatic polymerization of aniline was first performed in lignosulfonate (LGS) template system. High-redox-potential catalyst laccase, isolated from Aspergillus, was used as a biocatalyst in the synthesis of conducting polyaniline/lignosulfonate (PANI-ES-LGS) complex using atmospheric oxygen as the oxidizing agent. The linear templates (LGS), also serving as the dopants, could facilitate the directional alignment of the monomer and improve the solubility of the conducting polymer. The process of the polymerization was monitored using UV-Vis spectroscopy, by which the conditions for laccase-catalyzed synthesis of PANI-ES-LGS complex were also optimized. The structure characterizations and solubility of the complex were carried out using corresponding characterization techniques respectively. The PANI-ES-LGS suspensions obtained was used as coating for cotton with a conventional padder to explore the applications of the complex. The variable optoelectronic properties of the coated cotton were confirmed by cyclic voltammetry and color strength test. The molecular weight changes of LGS treated by laccase were also studied to discuss the mechanism of laccase catalyzed aniline polymerization in LGS template system.

Ultrasound enhances lipase-catalyzed synthesis of poly (ethylene glutarate)

The present work explores the best conditions for the enzymatic synthesis of poly (ethylene glutarate) for the first time. The start-up materials are the liquids; diethyl glutarate and ethylene glycol diacetate, without the need of addition of extra solvent. The reactions are catalyzed by lipase B from Candida antarctica immobilized on glycidyl methacrylate-ter-divinylbenzene-ter-ethylene glycol dimethacrylate at 40 °C during 18 h in water bath with mechanical stirring or 1 h in ultrasonic bath followed by 6 h in vacuum in both the cases for evaporation of ethyl acetate. The application of ultrasound significantly intensified the polyesterification reaction with reduction of the processing time from 24 to 7 h. The same degree of polymerization was obtained for the same enzyme loading in less time of reaction when using the ultrasound treatment. The degree of polymerization for long-term polyesterification was improved approximately 8-fold due to the presence of sonication during the reaction. The highest degree of polymerization achieved was 31, with a monomer conversion of 96.77%. The ultrasound treatment demonstrated to be an effective green approach to intensify the polyesterification reaction with enhanced initial kinetics and high degree of polymerization.

Metabolic engineering with multi-objective optimization of kinetic models

Kinetic models have a great potential for metabolic engineering applications. They can be used for testing which genetic and regulatory modifications can increase the production of metabolites of interest, while simultaneously monitoring other key functions of the host organism. This work presents a methodology for increasing productivity in biotechnological processes exploiting dynamic models. It uses multi-objective dynamic optimization to identify the combination of targets (enzymatic modifications) and the degree of up- or down-regulation that must be performed in order to optimize a set of pre-defined performance metrics subject to process constraints. The capabilities of the approach are demonstrated on a realistic and computationally challenging application: a large-scale metabolic model of Chinese Hamster Ovary cells (CHO), which are used for antibody production in a fed-batch process. The proposed methodology manages to provide a sustained and robust growth in CHO cells, increasing productivity while simultaneously increasing biomass production, product titer, and keeping the concentrations of lactate and ammonia at low values. The approach presented here can be used for optimizing metabolic models by finding the best combination of targets and their optimal level of up/down-regulation. Furthermore, it can accommodate additional trade-offs and constraints with great flexibility.

SMYD3 contributes to a more aggressive phenotype of prostate cancer and targets Cyclin D2 through H4K20me3

Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa.

Synthesis of new peptide derivatives that self-assemble into nanostructured hydrogels for biomedical applications

Tese de Doutoramento em Ciências (área de especialização em Química)

Presence of starch enhances in vitro biodegradation and biocompatibility of a gentamicin delivery formulation

The effect of α-amylase degradation on the release of gentamicin from starch-conjugated chitosan microparticles was investigated up to 60 days. Scanning electron microscopic observations showed an increase in the porosity and surface roughness of the microparticles as well as reduced diameters. This was confirmed by 67% weight loss of the microparticles in the presence of α-amylase. Over time, a highly porous matrix was obtained leading to increased permeability and increased water uptake with possible diffusion of gentamicin. Indeed, a faster release of gentamicin was observed with α-amylase. Starch-conjugated chitosan particles are non-toxic and highly biocompatible for an osteoblast (SaOs-2) and fibroblast (L929) cell line as well as adipose-derived stem cells. When differently produced starch-conjugated chitosan particles were tested, their cytotoxic effect on SaOs-2 cells was found to be dependent on the crosslinking agent and on the amount of starch used.

Optimization of low-cost nutritional supplementation for lignocellulosic ethanol fermentation

[Excerpt] Bioethanol from lignocellulosic materials (LCM), also called second generation bioethanol, is considered a promising alternative to first generation bioethanol. An efficient production process of lignocellulosic bioethanol involves an effective pretreatment of LCM to improve the accessibility of cellulose and thus enhance the enzymatic saccharification. One interesting approach is to use the whole slurry from treatment, since allows economical and industrial benefits: washing steps are avoided, water consumption is lower and the sugars from liquid phase can be used, increasing ethanol concentration [1]. However, during the pretreatment step some compounds (such as furans, phenolic compounds and weak acids) are produced. These compounds have an inhibitory effect on the microorganisms used for hydrolysate fermentation [2]. To overcome this, the use of a robust industrial strain together with agro-industrial by-products as nutritional supplementation was proposed to increase the ethanol productivities and yields. (...)

An efficient Azorean thermophilic consortium for lignocellulosic biomass degradation

[Excerpt] Lignocellulosic plant biomass is being envisioned by biorefinery industry as an alternative to current petroleum platform because of the large scale availability, low cost and environmentally benign production. The industrial bioprocessing designed to transform lignocellulosic biomass into biofuels are harsh and the enzymatic reactions may be severely compromised reducing the production of fermentable sugars from lignocellulosic biomass. Thermophilic bacteria consortium are a potential source of cellulases and hemicellulases adapted to extreme environmental conditions, which can be exploited as a new source for the development of more robust enzymatic cocktails. (...)

Bio-detoxification of mycotoxins by lactic acid bacteria from different food matrices

Lactic acid bacteria (LAB) play a key role in the biopreservation of a wide range of fermented food products, such as yogurt, cheese, fermented milks, meat, fish, vegetables (sauerkraut, olives and pickles), certain beer brands, wines and silage, allowing their safe consumption, which gave to these bacteria a GRAS (Generally Recognised as Safe) status. Besides that, the use of LAB in food and feed is a promising strategy to reduce the exposure to dietary mycotoxins, improving their shelf life and reducing health risks, given the unique mycotoxin decontaminating characteristic of some LAB. Mycotoxins present carcinogenic, mutagenic, teratogenic, neurotoxic and immunosuppressive effects over animals and Humans, being the most important ochratoxin A (OTA), aflatoxins (AFB1), trichothecenes, zearalenone (ZEA), fumonisin (FUM) and patulin. In a previous work of our group it was observed OTA biodegradation by some strains of Pediococcus parvulus isolated from Douro wines. So, the aim of this study was to enlarge the screening of the biodetoxification over more mycotoxins besides OTA, including AFB1, and ZEA. This ability was checked in a collection of LAB isolated from vegetable (wine, olives, fruits and silage) and animal (milk and dairy products, sausages) sources. All LAB strains were characterized phenotypically (Gram, catalase) and genotypically. Molecular characterisation of all LAB strains was performed using genomic fingerprinting by MSP- PCR with (GTG)5 and csM13 primers. The identification of the isolates was confirmed by 16S rDNA sequencing. To study the ability of LAB strains to degrade OTA, AFB1 and ZEA, a MRS broth medium was supplemented with 2.0 g/mL of each mycotoxin. For each strain, 2 mL of MRS supplemented with the mycotoxins was inoculated in triplicate with 109 CFU/mL. The culture media and bacterial cells were extracted by the addition of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v) to the culture tubes. A 2 mL sample was then collected and filtered into a clean 2 mL vial using PP filters with 0.45 m pores. The samples were preserved at 4 °C until HPLC analysis. Among LAB tested, 10 strains isolated from milk were able to eliminate AFB1, belonging to Lactobacillus casei (7), Lb. paracasei (1), Lb. plantarum (1) and 1 to Leuconostoc mesenteroides. Two strains of Enterococcus faecium and one of Ec. faecalis from sausage eliminated ZEA. Concerning to strains of vegetal origin, one Lb. plantarum isolated from elderberry fruit, one Lb. buchnerii and one Lb. parafarraginis both isolated from silage eliminated ZEA. Other 2 strains of Lb. plantarum from silage were able to degrade both ZEA and OTA, and 1 Lb. buchnerii showed activity over AFB1. These enzymatic activities were also verified genotypically through specific gene PCR and posteriorly confirmed by sequencing analysis. In conclusion, due the ability of some strains of LAB isolated from different sources to eliminate OTA, AFB1 and ZEA one can recognize their potential biotechnological application to reduce the health hazards associated with these mycotoxins. They may be suitable as silage inoculants or as feed additives or even in food industry.

Role of sirtuin 3 on mitochondrial dynamics in Huntington's disease striatal cells

Dissertação de mestrado em Genética Molecular

Compositional analysis of biofilms formed by Staphylococcus aureus isolated from food sources

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.00390

In vitro phosphorylation as tool for modification of silk and keratin fibrous materials

An overview is given of the recent work on in vitro enzymatic phosphorylation of silk fibroin and human hair keratin. Opposing to many chemical "conventional" approaches, enzymatic phosphorylation is in fact a mild reaction and the treatment falls within "green chemistry" approach. Silk and keratin are not phosphorylated in vivo, but in vitro. This enzyme-driven modification is a major technological breakthrough. Harsh chemical chemicals are avoided, and mild conditions make enzymatic phosphorylation a real "green chemistry" approach. The current communication presents a novel approach stating that enzyme phosphorylation may be used as a tool to modify the surface charge of biocompatible materials such as keratin and silk.