MMP-9/RECK imbalance: a mechanism associated with high-grade cervical lesions and genital infection by Human Papillomavirus (HPV) and Chlamydia trachomatis

"Manuscript"

Synthesis of iminosugars through diastereo/enantioselective diels-alder cycloaddition

Tese de Doutoramento em Ciências (Especialidade em Química)

Hydrocarbonoclastic bacteria: from bioremediation to bioenergy feedstock

Tese de Doutoramento em Engenharia Química e Biológica.

Bacteriophage-encoded depolymerases: their diversity and biotechnological applications

Bacteriophages (phages), natural enemies of bacteria, can encode enzymes able to degrade polymeric substances. These substances can be found in the bacterial cell surface, such as polysaccharides, or are produced by bacteria when they are living in biofilm communities, the most common bacterial lifestyle. Consequently, phages with depolymerase activity have a facilitated access to the host receptors, by degrading the capsular polysaccharides, and are believed to have a better performance against bacterial biofilms, since the degradation of extracellular polymeric substances by depolymerases might facilitate the access of phages to the cells within different biofilm layers. Since the diversity of phage depolymerases is not yet fully explored, this is the first review gathering information about all the depolymerases encoded by fully sequenced phages. Overall, in this study, 160 putative depolymerases, including sialidases, levanases, xylosidases, dextranases, hyaluronidases, peptidases as well as pectate/pectin lyases, were found in 143 phages (43 Myoviridae, 47 Siphoviridae, 37 Podoviridae, and 16 unclassified) infecting 24 genera of bacteria. We further provide information about the main applications of phage depolymerases, which can comprise areas as diverse as medical, chemical, or food-processing industry.

Combined bioremediation and enzyme production by Aspergillus sp. in olive mill and winery wastewaters

Olive mill wastewaters (OMW) and vinasses (VS) are effluents produced respectively by olive mills and wineries, both sectors are of great economic importance in Mediterranean countries. These effluents cause a large environmental impact, when not properly processed, due to their high concentration of phenolic compounds, COD and colour. OMW may be treated by biological processes but, in this case, a dilution is necessary, increasing water consumption. The approach here in proposed consists on the bioremediation of OMW and VS by filamentous fungi. In a screening stage, three fungi (Aspergillus ibericus, Aspergillus uvarum, Aspergillus niger) were selected to bioremediate undiluted OMW, two-fold diluted OMW supplemented with nutrients, and a mixture of OMW and VS in the proportion 1:1 (v/v). Higher reductions of phenolic compounds, colour and COD were achieved mixing both residues; with A. uvarum providing the best results. In addition, the production of enzymes was also evaluated during this bioremediation process, detecting in all cases lipolytic, proteolytic and tannase activities. A. ibericus, A. uvarum and A. niger achieved the highest value of lipase (1253.7 ± 161.2 U/L), protease (3700 ± 124.3 U/L) and tannase (284.4 ± 12.1 U/L) activities, respectively. Consequently, this process is an interesting alternative to traditional processes to manage these residues, providing simultaneously high economic products, which can be employed in the same industries.

Promising blood-derived biomarkers for estimation of the postmortem interval

A precise estimation of the postmortem interval (PMI) is one of the most important topics in forensic pathology. However, the PMI estimation is based mainly on the visual observation of cadaverous pheno- mena (e.g. algor, livor and rigor mortis) and on alternative methods such as thanatochemistry that remain relatively imprecise. The aim of this in vitro study was to evaluate the kinetic alterations of several bio- chemical parameters (i.e. proteins, enzymes, substrates, electrolytes and lipids) during putrefaction of human blood. For this purpose, we performed kinetic biochemical analysis during a 264 hour period. The results showed a significant linear correlation between total and direct bilirubin, urea, uric acid, transferrin, immunoglobulin M (IgM), creatine kinase (CK), aspartate transaminase (AST), calcium and iron with the time of blood putrefaction. These parameters allowed us to develop two mathematical models that may have predictive values and become important complementary tools of traditional methods to achieve a more accurate PMI estimation

Glucose addiction in cancer therapy : advances and drawbacks

Glucose addiction in cancer therapy: advances and drawbacks.

Pharmacological evaluation of plant extracts from Chapada Diamantina (Bahia, Brazil): Focus on antioxidant and cytotoxic properties

Tese de Doutoramento em Biologia de Plantas MAP - Bioplant

Synthetic biology approaches to engineer new pathways for the production of plant secondary metabolites

Secondary metabolites from plants are important sources of high-value chemicals, many of them being pharmacologically active. These metabolites are commonly isolated through inefficient extractions from natural biological sources and are often difficult to synthesize chemically. Therefore, their production using engineered organisms has lately attracted an increased attention. Curcuminoids, an example of such metabolites, are produced in Curcuma longa and exhibit anti-cancer and anti-inflammatory activities. Herein we report the construction of an artificial biosynthetic pathway for the curcuminoids production in Escherichia coli. Different 4-coumaroyl-CoA ligases (4CL) and polyketide synthases (diketide-CoA synthase (DCS), curcumin synthase (CURS) and curcuminoid synthase) were tested. The highest curcumin production (70 mg/L) was obtained by feeding ferulic acid and with the Arabidopsis thaliana 4CL1 and C. longa DCS and CURS enzymes. Other curcuminoids (bisdemethoxy- and demethoxycurcumin) were also produced by feeding coumaric acid or a mixture of coumaric and ferulic acids, respectively. Curcuminoids, including curcumin, were also produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase and 4-coumarate 3-hydroxylase were used. Caffeoyl-CoA O-methyltransferase was used to convert caffeoyl-CoA to feruloyl-CoA. This pathway represents an improvement of the curcuminoids heterologous production. The construction of this pathway in another model organism is being considered, as well as the introduction of alternative enzymes.

Biological extraction of bromelain from pineapple byproducts

[Excerpt] Isolation and purification of valuable compounds are very important processes to valorize agro-food byproducts. Currently, protein extraction and development of environmentally friendly technologies are industrially relevant topics [1]. Among the extracted proteins from byproducts proteases are a relevant group for industrial applications. These enzymes are a class of hydrolytic enzymes capable of cleaving the peptide bonds of proteins chains and are essential in physiological processes [2]. (...)

New bio-strategies for ochratoxin A detoxification using lactic acid bacteria

The occurrence of mycotoxigenic moulds such as Aspergillus, Penicillium and Fusarium in food and feed has an important impact on public health, by the appearance of acute and chronic mycotoxicoses in humans and animals, which is more severe in the developing countries due to lack of food security, poverty and malnutrition. This mould contamination also constitutes a major economic problem due the lost of crop production. A great variety of filamentous fungi is able to produce highly toxic secondary metabolites known as mycotoxins. Most of the mycotoxins are carcinogenic, mutagenic, neurotoxic and immunosuppressive, being ochratoxin A (OTA) one of the most important. OTA is toxic to animals and humans, mainly due to its nephrotoxic properties. Several approaches have been developed for decontamination of mycotoxins in foods, such as, prevention of contamination, biodegradation of mycotoxins-containing food and feed with microorganisms or enzymes and inhibition or absorption of mycotoxin content of consumed food into the digestive tract. Some group of Gram-positive bacteria named lactic acid bacteria (LAB) are able to release some molecules that can influence the mould growth, improving the shelf life of many fermented products and reducing health risks due to exposure to mycotoxins. Some LAB are capable of mycotoxin detoxification. Recently our group was the first to describe the ability of LAB strains to biodegrade OTA, more specifically, Pediococcus parvulus strains isolated from Douro wines. The pathway of this biodegradation was identified previously in other microorganisms. OTA can be degraded through the hydrolysis of the amide bond that links the L-β-phenylalanine molecule to the ochratoxin alpha (OTα) a non toxic compound. It is known that some peptidases from different origins can mediate the hydrolysis reaction like, carboxypeptidase A an enzyme from the bovine pancreas, a commercial lipase and several commercial proteases. So, we wanted to have a better understanding of this OTA degradation process when LAB are involved and identify which molecules where present in this process. For achieving our aim we used some bioinformatics tools (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV). We also designed specific primers and realized gene specific PCR. The template DNA used came from LAB strains samples of our previous work, and other DNA LAB strains isolated from elderberry fruit, silage, milk and sausages. Through the employment of bioinformatics tools it was possible to identify several proteins belonging to the carboxypeptidase family that participate in the process of OTA degradation, such as serine type D-Ala-D-Ala carboxypeptidase and membrane carboxypeptidase. In conclusions, this work has identified carboxypeptidase proteins being one of the molecules present in the OTA degradation process when LAB are involved.

Microbial respiration with chlorine oxyanions: diversity and physiological and biochemical properties of chlorate- and perchlorate-reducing microorganisms

Chlorine oxyanions are valuable electron acceptors for microorganisms. Recent findings have shed light on the natural formation of chlorine oxyanions in the environment. These suggest a permanent introduction of respective compounds on Earth, long before their anthropogenic manufacture. Microorganisms that are able to grow by the reduction of chlorate and perchlorate are affiliated with phylogenetically diverse lineages, spanning from the Proteobacteria to the Firmicutes and archaeal microorganisms. Microbial reduction of chlorine oxyanions can be found in diverse environments and different environmental conditions (temperature, salinities, pH). It commonly involves the enzymes perchlorate reductase (Pcr) or chlorate reductase (Clr) and chlorite dismutase (Cld). Horizontal gene transfer seems to play an important role for the acquisition of functional genes. Novel and efficient Clds were isolated from microorganisms incapable of growing on chlorine oxyanions. Archaea seem to use a periplasmic Nar-type reductase (pNar) for perchlorate reduction and lack a functional Cld. Chlorite is possibly eliminated by alternative (abiotic) reactions. This was already demonstrated for Archaeoglobus fulgidus, which uses reduced sulfur compounds to detoxify chlorite. A broad biochemical diversity of the trait, its environmental dispersal, and the occurrence of relevant enzymes in diverse lineages may indicate early adaptations of life toward chlorine oxyanions on Earth.

An overview of the brewing process

The first chapter of this book has an introductory character, which discusses the basics of brewing. This includes not only the essential ingredients of beer, but also the steps in the process that transforms the raw materials (grains, hops) into fermented and maturated beer. Special attention is given to the processes involving an organized action of enzymes, which convert the polymeric macromolecules present in malt (such as proteins and polysaccharides) into simple sugars and amino acids; making them available/assimilable for the yeast during fermentation.

Thermal technologies in food processing

[Excerpt] Introduction: Thermal processing is probably the most important process in food industry that has been used since prehistoric times, when it was discovered that heat enhanced the palatability and the life of the heat-treated food. Thermal processing comprehends the heating of foods at a defined temperature for a certain length of time. However, in some foods, the high thermotolerance of certain enzymes and microorganisms, their physical properties (e.g.,highviscosity),ortheircomponents(e.g.,solidfractions) require the application of extreme heat treatments that not only are energy intensive, but also will adversely affect the nutritional and organoleptic properties of the food. Technologies such as ohmic heating, dielectric heating (which includes microwave heating and radiofrequency heating), inductive heating, and infrared heating are available to replace, or complement, the traditional heat-dependent technologies (heating through superheated steam, hot air, hot water, or other hot liquid, being the heating achieved either through direct contact with those agents – mostly superheated steam – or through contact with a hot surface which is in turn heated by such agents). Given that the “traditional” heatdependent technologies are thoroughly described in the literature, this text will be mainly devoted to the so-called “novel” thermal technologies. (...)

Enzymatic biofinishes for synthetic textiles

Fundação para a Ciência e a Tecnologia, Grant SFRH/BPD/46515/2008. Jiajia Fu acknowledges the support of Jiangsu Provincial Natural Science Foundation of China (No. BK2012112) and the National Natural Science Foundation of China under Grant No. 3120113