Faculdade de Ciências
Permanent URI for this community
Browse
Browsing Faculdade de Ciências by Field of Science and Technology (FOS) "Ciências Naturais::Outras Ciências Naturais"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
- Avaliação da atividade biológica de compostos naturais de plantas medicinais de Angola e de compostos de síntesePublication . Fernandes, Nelson António Freitas; Mendonça, António José Geraldes de; Mendonça, Dina Isabel Malheiros Dinis deNeste trabalho foram avaliadas as atividades antioxidante, antimicrobiana e de inibição da acetilcolinesterase para 8 compostos de síntese, 4 compostos naturais e 50 extratos obtidos com diferentes solventes a partir de 16 plantas recolhidas em Angola (Hymenodictyon floribundum; Parinari capensis; Tinnea antiscorbutica; Eragrostis viscosa; Xylopia odoratissima; Peucedanum angolense; Boscia microphylla; Adenodolichos huillensis; Rhus kirkii; Croton gratisssimus; Gymnosporia senegalensis; Solanecio mannii; Peltophorum africanum; Piliostigma thonningii; Phragmanthera glaucocarpa e Solanum incanum). O estudo da capacidade antioxidante foi realizado pelos métodos de ABTS, DPPH e valor peróxido. A concentração de compostos fenólicos foi determinada pelo método de Folin-Ciocalteu e a inibição da acetilcolinesterase pelos métodos de Ellman e bioautográfico de cromatografia em camada fina. A atividade antimicrobiana foi estudada pelas técnicas de microdiluição e de difusão em disco. O extrato de ramos de P. africanum em acetato de etilo foi o que apresentou uma concentração mais elevada de compostos fenólicos totais (746,56 ± 0,05 mg G.A.E./g extrato). O extrato aquoso das folhas de P. capensis foi o que apresentou melhor reatividade contra o radical ABTS (EC50 de 0,57 ± 0,03 mg/mL), e o extrato aquoso das raízes de P. glaucocarpa foi o que apresentou melhor reatividade contra o radical DPPH (EC50 de 0,10 ± 0,01 mg/mL). Em relação ao valor peróxido o extrato aquoso das folhas de A. huillensis foi o que mais inibiu a formação de peróxidos (34,47 ± 1,20 meq O2/g de amostra) a partir de óleo de girassol após oito dias em condições de oxidação. Os resultados obtidos para a atividade antimicrobiana demonstram que os extratos e compostos de síntese não possuem atividade contra o S. aureus ATCC 25923, a E. coli ATCC 25922 e a C. albicans ATCC 24433. Em relação à inibição da enzima acetilcolinesterase o melhor resultado foi obtido para o extrato metanólico dos ramos de P. africanum (IC50 0,23 ± 0,00 mg/mL). Os compostos isolados dos extratos vegetais possuem pouca atividade antioxidante (EC50 alto). Em relação á inibição da acetilcolinesterase os compostos isolados dos extratos vegetais possuem também uma baixa capacidade de inibição da enzima (IC50 alto). Tendo em conta os resultados obtidos a P. capensis e P. africanum podem ser consideradas uma fonte promissora de novos compostos com ação de inibição da acetilcolinesterase e capacidade antioxidante.
- Biosynthesis, isolation and kinetic characterization of recombinant human catechol-O-methyltransferase from Pichia pastoris strainsPublication . Pedro, Augusto Quaresma Henriques; Passarinha, Luís António Paulino; Queiroz, João António de Sampaio RodriguesCatechol-O-methyltransferase (COMT; EC 2.1.1.6) is a magnesium-dependent enzyme that catalyzes the methylation reaction of different catecholic substrates such as catecholamines, xenobiotic catechols and catecholestrogens. Following the initial characterizations of these enzymes, it was described that they are potentially involved in diverse human disorders. Specifically, as its inhibition has proven to be of great interest in neurologic disorders such as Parkinson's disease, developing inhibitor molecules with increased potency and selectivity may improve the outcome of these patients. These molecules are usually accomplished using structure-based drug design studies that rely on the attainment of highly purified protein quantities. Indeed, challenges in the determination of protein structures are mainly associated with their low natural abundance coupled with the difficulty of obtaining crystals amenable to X-Ray diffraction. In particular, as membrane proteins are naturally embedded in the lipid bilayer, the determination of their structure faces additional difficulties. As it is unrealistic to purify all of these targets from their natural sources, structural biology of proteins usually focus on the recombinant heterologous expression of these proteins onto an expression host. In addition, to isolate the target proteins from the other major host contaminants, equally appropriated purification strategies need to be designed and implemented, mostly using chromatographic procedures. Throughout this entire process, is also important that the developed strategy is able to keep the proteins in a stable and functional active form, thus avoiding its misfolding during biosynthesis and aggregation after its recovery and isolation in the downstream processing. Therefore, the main scope of this work is the development of a straightforward approach that allows the biosynthesis, isolation and purification of recombinant human COMT isoforms in a biologically active form for further application in structural studies or to evaluate their role as potential therapeutic proteins. Specifically, although no single host can provide all the desired properties for recombinant protein biosynthesis, Pichia pastoris is able to perform many post-translational modifications and is cultivated at high cell-densities in moderately cheap media. Therefore, in this work, it was selected for expression of COMT enzymes. On the other hand, the high selectivity often provided by affinity chromatography prompted us to employ it as the main isolation and purification step. The determination of COMT enzymatic activity is greatly important in COMT recombinant research, either to assess COMT activity from recombinant lysates or purified fractions, for detergent-solubilized or unsolubilized samples and for both isoforms. Therefore, a faster and more sensitive analytical method based on HPLC coupled with coulometric detection was developed for quantifying metanephrine in these assays. Then, an integrated strategy for recombinant soluble catechol-O-methyltransferase (SCOMT) biosynthesis onto P. pastoris and purification using immobilized-metal affinity chromatography was implemented where highly purified fractions of this target enzyme were obtained. On the other hand, as heterologous membrane protein overexpression is usually more challenging than soluble proteins and less reports are available in the literature with recombinant human membrane-bound catechol-O-methyltransferase (MBCOMT) than COMT soluble isoform, our work were mostly focused on MBCOMT. Here, we established protocols for MBCOMT expression in Pichia pastoris methanol-induced cultures in baffled shake-flasks and mini-bioreactors. In particular, the optimization of the induction phase using artificial neural networks in mini-biorreactors allowed achieving high levels of biologically active MBCOMT. Then, arginine-affinity chromatography was successfuly applied for the direct capture of MBCOMT from Pichia pastoris lysates and it was recovered in a moderate purified form. Finally, the ongoing work is related to the purification of a hexa-histidine tagged form of MBCOMT using immobilized-metal affinity chromatography. Indeed, despite significant achievements were made concerning the construction of a tagged form of MBCOMT solubilized with an appropriated detergent in a biologically active form, additional stepwise gradients are required to effectively separate MBCOMT from the other contaminants. In conclusion, the progress achieved with this work meets the highly demanding requirements of biophysical techniques, mainly regarding the upstream stage as well as COMT stabilization where moderate to high quantities of catalitically active enzymes were obtained. In particular, coupling the strategy here reported for SCOMT with a final polishing step will probably allow performing structural or bio-interaction studies with this enzyme. Nonetheless, the strategies here described successfully for partial MBCOMT purification need to be improved, especially for immobilized-metal affinity chromatography once it is considered to be highly selective and, thus, it is feasible that after succesful optimization procedures, fractions with high purity will be obtained. Therefore, the strategies here reported with the intensification and optimization of some procedures would possible permit performing structural and bio-interaction studies using the apo-enzymes or complexed with different ligands (cofactors or inhibitors) by Nuclear Magnetic Ressonance, Isothermal Titration Calorimetry or even using Crystallographic experiments.
- Evaluation of Alt a 1 as specific marker of exposure to fungal allergenic sources and clinical relevance of a manganese-dependent superoxide dismutase and a serine protease as new Alternaria alternata allergensPublication . Gabriel, Marta Sofia da Fonseca; Tomaz, Cândida Ascensão Teixeira; Marcos, Jorge MartinezAllergic diseases are considered to be one of the epidemics of the century, affecting approximately one-third of the general population. Classically, among the allergenic sources able to induce IgE-mediated reactions, fungi have been one of the less favored areas of study. It has been demonstrated that sensitization to fungal aeroallergens, particularly from Alternaria alternata, represents an unequivocal risk factor for the development, persistence and severity of asthma. Thus, the better understanding and management of fungal allergy, namely by improvements in the actual assessment and diagnostic approaches are needed. Regarding the assessment of fungal exposure, actual methodologies are generally laborious and time-consuming making difficult to establish or exclude a fungal contamination and potential associations with allergic disease. In terms of diagnosis, the main difficulty arises from the high number of patients that are apparently sensitized to multiple fungi in which the identification of primary cause of sensitization is complex. Because A. alternata is one of the most abundant and potent source of airborne allergens, the panel of allergenic components produced by this fungal specie, seems to be a very relevant target of study. Among the several allergens described in this mold, Alt a 1 has been demonstrated to be the most important, sensitizing approximately 80% of A. alternata allergic patients. For this reason, Alt a 1 has been used as the diagnostic marker of genuine sensitization to A. alternata. However, given the complex A. alternata sensitization data, which shows a significant level of polysensitization, due to co-sensitization and/or cross-reactivity to several other phylogenetically related and non-related molds, other allergenic A. alternata components should be studied to explain the whole broad range of reported clinical observations. In the last years, componentresolved diagnosis (CRD) has been shown to be a valuable tool to elucidate clinical observations and to differentiate between cases of co-sensitization and cross-reactivity. Nevertheless, the actual available panel of A. alternata allergens appears to not be enough for achieving an accurate diagnosis and prognosis of sensitization to this mold. Considering the above mentioned facts, the major aims of this work were, on one hand to develop an approach to specifically detect Alternaria and related species by using the A. alternata major allergen, Alt a 1, as a specie-specific molecular marker. And, on other hand, to characterize two new cross-reactive A. alternata allergens belonging to the manganesedependent superoxide dismutase (MnSOD) and serine protease (SP) protein families and to study their role in A. alternata sensitization. The strategies used to accomplish both aims made use of phylogenetic relationships among fungal species that share A. alternata allergen homologues. First, investigating conservation of the genes encoding for Alt a 1 and its homologues which allowed the design of a set of primers in the conserved immunologically relevant Alt a 1 coding sequence region. This primer set, together with a pair of primers to amplify the complete Alt a 1 encoding gene, was applied in a polymerase chain reaction (PCR)-based system. This approach yielded two rapid, sensitive and specific methodologies with high potential to be applied both for the detection of Alt a 1 and Alt a 1 homologues and for specific identification of the existence of contamination by the very close taxonomically related species A. alternata and A. tenuissima. The investigation of conservation of the genes encoding for A. alternata protein homologues, was also employed to design primers in the invariant region of fungal MnSOD and SP nucleotide sequences available in public databases. The aforementioned primers along with rapid amplification of cDNA ends (RACE) and sequencing assays allowed for the isolation of the full-length cDNA encoding for A. alternata MnSOD and SP. Both proteins were then produced as recombinant proteins in E. coli and evaluated for IgE immunoreactivity using a comprehensive panel of sera from patients clinically labeled as to be sensitized to A. alternata. Immunoblotting analysis showed that IgE antibodies from A. alternata-sensitized patients bound to recombinant A. alternata MnSOD and SP with prevalence of 11.5% (n=61) and 10.2% (n=59), respectively. These results were reported to the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub- Committee, and both proteins, respectively named Alt a 14 and Alt a 15, are currently included in the official A. alternata allergen list. By performing immunoblotting inhibition assays it was demonstrated that Alt a 14 and Alt a 15 are able to mediate IgE cross-reactivity with similar homologues allergens from other important allergenic fungal species, such as A. fumigatus and C. lunata, respectively. Furthermore, evidence of reactivity of Alt a 14 to IgE of Allergic Bronchopulmonary Aspergillosis (ABPA)-diagnosed patients was found, thus indicating that sensitization to this A. alternata allergen could play important implications in the development of ABPA. On the other hand, sensitization to Alt a 15 was shown to be restricted to apparently poly-sensitized patients and to justify some cases of sensitization to A. alternata in which there is no evidence of Alt a 1 sensitization. A homologue to Alt a 14, together with a manitol desidrogenase (MtDH), of the edible mushroom A. bisporus were identified to induce an anaphylactic shock reaction in a patient who presented a previous history of respiratory allergic symptoms associated to mold aeroallergens. These results were useful in proving that although minor allergens, A. alternata cross-reacting proteins may be the primary cause of strong allergic reactions to various other allergenic sources, such as mushrooms. Overall, in this work we successfully developed a specific and sensitive PCR method based on the amplification of regions of the gene encoding for the allergenic Alt a 1 and Alt a 1 homologues which it is intended to be applied for environmental monitoring as well as for quality and biosecurity control of food stuffs. Moreover, cloning and characterization of Alt a 14 and Alt a 15 as minor allergens of A. alternata that can trigger cross-reactive IgE response with other important and prevalent allergenic fungal species were also accomplished. The availability of these allergens as recombinant molecules suitable for application in a molecule-based diagnostic approach to fungal allergy can improve the diagnostic process prognosis of clinical manifestations and potential cross-reactivities. Furthermore, this can guide to a more effective specific immunotherapy using a single or a few allergenic molecules. Hence, this work provided valuable findings that can contribute to improving the accuracy of assessment of allergen exposure, diagnosis and management of IgE-mediated fungal diseases.
- miRNA-29 bioseparation and target delivery strategies for Alzheimer's diseasePublication . Pereira, Patrícia Alexandra Nunes; Sousa, Fani Pereira de; Figueiras, Ana Rita; Correia, Ilídio Joaquim SobreiraThe possibility of selectively alter the expression pattern of a particular gene has been sought by scientists and clinicians for a long time. Nowadays, RNA interference (RNAi)-based technology has become a novel tool for silencing gene expression in cells. In addition, this strategy encloses an enormous therapeutic potential that could change the course of the currently applied treatments in several life threatening pathologies and it is expected that this technology can be translated onto clinical applications in a near future. MicroRNA (miRNA) has become a commonly employed tool for gene silencing, since it prevents protein synthesis by inducing the messenger RNA (mRNA) degradation, with a high specificity degree. Consequently, in the last years, the miRNAs have emerged as biopharmaceuticals to regulate several pathways involved in the insurgence and progression of the Alzheimer’s disease (AD), since they might have key regulatory roles in many neuronal functions, such as differentiation, synaptic plasticity and memory formation, and typically they are down-regulated in disease conditions. In the literature there are some studies describing a causal relationship between miR-29 expression and AD, since a loss of miR-29 cluster can contribute to increased beta-amyloid precursor protein-converting enzyme 1 (BACE1) and Amyloid-β (Aβ) levels in sporadic AD patients. Thus, this evidence supports the possibility to use miR-29 as a potential therapeutic target for AD therapy. In general, miRNA-based therapy relies on the use of synthetic microRNAs. However, these synthesized formulations typically present contaminants that can lead to non-targeted gene silencing, which still restricts the pre-clinical or clinical application of these RNAs. Thus, considering this therapeutic purpose and the global distribution of novel biopharmaceuticals it is necessary to develop efficient processes for their preparation. The development of new strategies for microRNA production with high purity degree and biologically active is extremely required. One of the strategies might be the use of the recombinant production of biomolecules using prokaryotic hosts. Hence, the present work intends to develop and establish an integrative biotechnological platform to biosynthesize and purify a recombinant miRNA precursor (pre-miR-29b) to act in the selective silencing of endogenous pathways directly related with AD, in particular BACE1 and Aβ. In addition, the success of these therapies also depends upon the ability to selectively and efficiently deliver the pre-miR-29b in the cytoplasmic compartment of neuronal cells, the location where their function is exerted; therefore the development of miRNA delivery systems was also envisioned. The expression system Rhodovulum sulfidophilum (R. sulfidophilum) DSM 1374 allowed, for the first time, the production of human pre-miR-29b with a straightforward recuperation of pre-miR-29b in a single step, maintaining its biological active form. The application of this recombinant bacterial microorganism is innovative and is supported by the unusual capacity of secreting the nucleic acids to the extracellular space and the absence of host ribonucleases in the culture medium. Therefore, it is expected that the secreted miRNA will be devoid of main bacterial associated impurities. Regarding the growth conditions, and conversely to what was previously described for this bacterium, our results showed to be possible to develop an original approach for the aerobic growth of the R. sulfidophilum, which results in a cell growth improvement followed by an enhanced production of human pre-miR-29b. The extracellular pre-miR-29b concentration was approximately 182 μg/L, after 40 hours of bacterial growth and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 hours of cell growth. To further develop a potential therapeutic application, the major interest is not only to produce high quantities of RNA but also to obtain and preserve its biological active form, fulfilling the requirements of regulatory agencies. Hence, to assure that this prerequisite is met it was used a novel and effective purification strategy, based on affinity chromatography, to purify the pre-miR-29b. Therefore, in order to achieve the selectivity towards the target pre-miRNA and the maximum resolution between the pre-miR-29b and other host biomolecules (transfer RNAs and proteins) it was used an affinity support that exploits the same biological interactions that are established within the cell, by using immobilized amino acids (L-lysine and L-arginine), as specific ligands. The recognition of the pre-miR-29b achieved with these supports, allowed its selective recovery from a complex mixture with high efficiency and high purity. In parallel, the binding of pre-miRNA to these different amino acids was studied by Surface Plasmon Resonance. This information brings important insights concerning the characterization of the pre-miRNA binding onto chromatographic supports. Moreover, it was possible to determine some particular conditions enabling the improvement of the binding specificity of the amino acid ligands used to purify miRNA, preserving the RNA integrity. Taking into account that the structure of the chromatographic supports has been continuously developed to afford rapid and efficient separations, namely for the purification of nucleic acids, it was also tested a monolithic support to purify the pre-miR-29b. The association of the high capacity of these supports with the specificity conferred by the agmatine ligand (a derivative of L-arginine) represented a novelty and an advantage to obtain highly pure pre-miR-29b (90%) with a high recovery yield (95%). The establishment of an effective application of miRNAs is usually constrained by different phenomena, namely their easy degradation when in contact with the body fluids. To overcome this limitation, delivery systems, such as polymeric systems (polyplexes), were developed and characterized in order to encapsulate and protect the pre-miR-29b biopharmaceuticals from degradation, allowing their sustained and targeted release. The formulations prepared with chitosan and polyethylenimine demonstrated high loading capacity, small sizes and exhibited a strong positive charge on their surface. In addition, considering the application field of this work, the delivery systems should also have the ability to penetrate the Blood-Brain Barrier (BBB), causing an increase of the pre-miRNAs concentration in the brain and, consequently the improvement of the therapeutic effect. Actually, BBB is an intrinsic barrier limiting miRNA therapeutic effect on the central nervous system. Thus, to improve the delivery of pre-miRNA therapeutics in the brain, the polyplexes were functionalized with specific ligands, namely lactoferrin and stearic acid which are recognized by cell surface receptors of BBB. Finally, it was evaluated the biological activity of the recombinant pre-miR-29b by measuring the efficiency on human BACE1 knockdown, using in vitro neuronal cell lines. The effect of recombinant pre-miR-29b administration was verified by both assessing the mRNA and protein human BACE1 levels, by using RT-qPCR, Western blot and Imunocytochemistry. Results suggest that recombinant pre-miR-29b can represent a novel biopharmaceutical product for the therapeutic modulation of human BACE1 levels, because high levels of inhibition were achieved, namely 80% of reduction for BACE1 protein expression and 45% for Aβ42 levels. Globally, the implementation of these cutting-edge technologies can have a great impact on the biopharmaceutical industry, providing the basis for the implementation of novel miRNA-based therapeutics, not only for neurological disorders but also for future therapeutic targets that can be of potential interest.