Protein disulfide isomerase-assisted functionalization of proteinaceous substrates

Tese de doutoramento em Engenharia TêxtilThe formation of intramolecular disulphide bonds is critical in the process of protein folding and on the stabilization of protein tertiary structure. Their formation involves the oxidation of two thiol groups that should be correctly paired. The incorrect paring inhibit the protein folding into its native conformation. The rearrangement of incorrectly disulfide bonds on proteins is catalysed in vivo by the protein disulphide isomerase (PDI). This versatile enzyme is able to catalyse the oxidation, reduction and isomerisation of disulfide bonds in a broad range of protein substrates. This dissertation successfully presents the use of PDI for functionalization of cysteine-containing (CC) proteinaceous substrates such as keratin fibres and RNase A microspheres. These approaches take advantage of the presence of thiol moieties and disulphide bonds in these substrates. It was shown that the type of reaction catalysed by PDI can be predicted, by controlling the redox environment. When the active site of PDI is in its oxidized state due to is characteristic potential redox (Eº = -180 mV) an oxidation reaction is catalyzed. When the active site of PDI is transformed to its reduced state (ΔE = - 260 mV), the isomerisation of disulfide bonds is promoted. PDI was able to incorporate CC functional molecules on wool and hair trough disulfide bonds, as suggested by matrix-assisted laser desorption and ionization time-offlight results (MALDI-TOF) analysis. Similarly, PDI increased the affinity of a synthesised keratin-based peptide (KP) towards hair and facilitated the penetration into its cortex. Targeting a biomedical approach, Ribonuclease A (RNase A) was oxidatively attached to the wool surface through disulphide bonds, and PDI was shown to induce its release. Aiming a cosmetic application, KP and other synthesized surfactant-based peptide (SPB) were applied in over-bleached, damaged hair. Both peptides induced an improvement on its mechanical properties and thermal stability. Thus, recovering of the fibre integrity loss during the hair bleaching process was achieved. In the presence of PDI, peptides were linked by disulphide bonds and the thermal stability increased at higher levels. Due to the great properties developed on over-bleached hair after KP application, in the absence of PDI, other type of hair (relaxed hair) was treated with this peptide and the same properties were measured. The relaxing treatment, commonly applied on excessively curly hair, often result on the weakening of the fibre. KP was then applied to this weakened hair and its ability to recover its mechanical and thermal properties was proved. Two different peptide formulations were evaluated. In one formulation KP was dissolved in aqueous solution (WF) while in the other KP was dissolved in organic solvent solution (OF). The last imparted better mechanical and thermal properties to the hair, however, the safety assessment showed that OF is potentially cytotoxic, inhibit cell growth, and genotoxic. The KP itself did not inhibit the cell growth and was found to be non-cytotoxic and non-genotoxic, hence suitable for the application on cosmetic formulations at concentrations up to 0.5 g/L. Showing its ability to act on a broad range of cysteine-containing compounds, PDI was also able to oxidize the thiol groups found in sphere-like particles, recovering their biological function, at the conditions that lead PDI‟s active site on its oxidized state. Using a reducing environment, PDI promoted the released of native RNase A from protein-based microspheres. The research presented in this thesis shows the versatility of PDI to promote diverse functionalization on proteinaceous substrates, resulting in a wide applicability in different areas such as cosmetic, textile and biotechnology. The work was carried out partially in collaboration with a cosmetic company; hence the research included promising biotoolsbased strategies for hair-care product development, especially for the application on several types of damaged hair.As pontes dissulfídicas são uma característica muito importante para a estabilização da estrutura terciária de proteínas. A formação das mesmas envolve a oxidação de dois grupos tiol, os quais devem estar correctamente ligados. Caso contrário, a proteína perde a sua actividade biológica característica, devido à formação de pontes dissulfídicas nãonativas, e desnatura. In vivo, aquando da formação das proteínas na célula, uma enzima tem uma função muito importante: promover o rearranjo das pontes dissulfídicas nãonativas e prevenir a agregação das proteínas desnaturadas. Esta enzima é a Protein Disulfide Isomerase (PDI), uma isomerase de pontes dissulfídicas que catalisa a oxidação, redução ou isomerisação de uma vasta gama de substratos contendo cisteína na sua constituição. Esta dissertação apresenta com sucesso a aplicação desta enzima na funcionalização substratos proteicos, tais como as fibras queratinosas ou microesferas de Ribonuclease A (RNase A). As pontes dissulfídicas dos substratos, acima mencionados, são as ligações-alvo nesta abordagem. Foi demonstrado que o potencial de redução do centro activo da PDI é uma ferramenta importante na pré-determinação das reacções que esta enzima pode catalisar. Quando o centro activo da PDI está no seu estado oxidado, devido ao seu característico potencial redox (Eº = -180 mV), uma reacção de oxidação é catalisada. Quando o centro activo da PDI é transformado para o seu estado reduzido (ΔE = - 260 mV), a isomerisação das pontes dissulfídicas é promovida. A PDI catalisou a incorporação de moléculas contendo cisteína (CC) em lã e cabelo através de pontes dissulfídicas, sugerido pela análise de espectroscopia de massa (MALDITOF). A PDI facilitou também, a penetração de um péptido de queratina (KP) no córtex da fibra de cabelo e induziu a libertação de uma proteína modelo (Ribonuclease A) da superfície da lã. Com o objectivo de uma aplicação cosmética, o KP e outro péptido, derivado de um surfactante humano (SPB) foram aplicados em cabelo danificado, previamente sujeito a vários ciclos de branqueamento oxidativo. Ambos os péptidos melhoraram as propriedades mecânicas e a estabilidade térmica do cabelo danificado, provando a sua capacidade para recuperar a integridade da fibra. Quando a PDI foi aplicada, os péptidos ligaram-se ao cabelo através de pontes dissulfídicas e a estabilidade térmica aumentou para valores ainda mais elevados. Devido ao efeito renovador que o KP teve sobre o cabelo branqueado, outro tipo de cabelo (cabelo relaxado) foi tratado com este péptido e as mesmas propriedades foram medidas. Os tratamentos de relaxamento em cabelo extremamente encaracolado resultam em enfraquecimento do mesmo. O KP foi por isso aplicado neste tipo de cabelo e a sua capacidade para o melhorar foi provado. Dois tipos de formulações peptídicas foram também testados. Numa formulação o KP foi diluído numa solução aquosa (WF), enquanto na outra o KP foi diluído numa solução contendo solventes orgânicos (OF). Esta última promoveu melhores resultados, contudo, revelou-se potencialmente cytotoxica, genotoxica e inibidora do crescimento celular. Demonstrou-se, todavia, que o péptido em solução aquosa pode ser aplicado em formulações cosméticas até à concentração de 0.5 g/L não manifestando citotóxicidade, genotóxicidade ou inibição do crescimento celular. Demonstrando a sua aptidão para actuar em compostos contendo cisteína, a PDI foi também capaz de oxidar os grupos tiol presentes nas microesferas, recuperando a sua função biológica nas condições que promovem o estado oxidado do seu centro activo. Usando um ambiente mais redutor, promoveu a libertação da proteína nativa das microesferas proteicas. Os resultados apresentados nesta tese demonstram a versatilidade da PDI para promover a funcionalização de substratos proteicos, resultando numa ampla aplicabilidade em áreas distintas como cosmética, têxtil e biotecnologia. O trabalho foi desenvolvido em parceria com uma empresa de cosmética, o que fomentou a procura de estratégias biológicas para o desenvolvimento de novos produtos para cabelo, especialmente para a aplicação em vários tipos de cabelo danificado.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/38363/200

Nanotransformation of vancomycin overcomes the intrinsic resistance of Gram-negative bacteria

The increased emergence of antibiotic-resistant bacteria is a growing public health concern, and although new drugs are constantly being sought, the pace of development is slow compared with the evolution and spread of multidrug- resistant species. In this study, we developed a novel broad-spectrum antimicrobial agent by simply transforming vancomycin into nanoform using sonochemistry. Vancomycin is a glycopeptide antibiotic largely used for the treatment of infections caused by Gram-positive bacteria but inefficient against Gram-negative species. The nanospherization extended its effect toward Gram-negative Escherichia coli and Pseudomonas aeruginosa, making these bacteria up to 10 and 100 times more sensitive to the antibiotic, respectively. The spheres were able to disrupt the outer membranes of these bacteria, overcoming their intrinsic resistance toward glycopeptides. The penetration of nanospheres into a Langmuir monolayer of bacterial membrane phospholipids confirmed the interaction of the nanoantibiotic with the membrane of E. coli cells, affecting their physical integrity, as further visualized by scanning electron microscopy. Such mechanism of antibacterial action is unlikely to induce mutations in the evolutionary conserved bacterial membrane, therefore reducing the possibility of acquiring resistance. Our results indicated that the nanotransformation of vancomycin could overcome the inherent resistance of Gram-negative bacteria toward this antibiotic and disrupt mature biofilms at antibacterial-effective concentrations.Peer ReviewedPostprint (author's final draft

Human stress during driving: correlation with road traffic externalities

The evaluation of vehicle occupants (VO) stress level is a research topic with recent and increasing interest as researchers pursue to understand its correlation with other road traffic-related externalities (RTE), i.e., pollutants and noise emissions, road safety and traffic congestion, and ultimately design a framework able to optimize road networks while considering road stress. Road singularities (e.g., roundabouts, traffic signal lights, and junctions) and even different road types (e.g., urban, rural, highways) may introduce distinct impacts on vehicle occupants (VO), i.e., regarding their emotional state and stress level. VO heart rate variability (HRV) presents a correlation with human stress and is usually applied to infer stress levels. It has been demonstrated the connection between driving styles and pollutants emissions as well as the likelihood of road crash occurrence, which in turn are associated with traffic congestion. The doctoral research main objective is to provide a framework to develop empirical multi-objective optimization and/or analysis of RTE considering stress based on vehicle and engine activity empirical data acquired through real-world driving or simulation. The main contributions from this research will be: 1) the study of the correlation of human stress with pollutant and noise emissions, road safety and traffic congestion through an integrated approach; 2) deliver an indicator, in the form of an equivalent monetary cost, accounting for human stress, pollutants and noise emissions, road safety and traffic congestion; 3) provide a method to assess human stress during driving without using any physiological equipment or data. These contributions will allow future road infrastructures and networks, and vehicle technology to be planned considering their human stress-related performance and consequently improve VO quality of life.publishe

Magneto-mechanically induced antimicrobial properties of cone-like shaped surfaces

Hygienic surfaces that prevent the proliferation of harmful microorganisms are required in a large variety of environments, including medical areas. Novel strategies are being developed to impede microorganisms colonization of surfaces. In this work, Terfenol-D cone-like shaped nanopatterned surfaces are fabricated by sputtering. The bactericidal effect of such surfaces owed to their morphology is increased in combination with an alternating magnetic field, which boosts the mechanical injury caused to the planktonic cells. Bactericidal assays with Gram-negative Escherichia coli are carried out under static (i.e. without any external stimuli) and dynamic (under the application of an alternating magnetic field) conditions for control silicon substrates, Terfenol-D films and nanostructured surfaces. The nanostructured surfaces at the dynamic condition exhibit the larger bactericidal effect. Bacterial adhesion on the materials was analyzed, and results show a reduction of the attachment surface of bacterial cells on Terfenol-D surfaces in comparison with the control silicon that are attributed both to material properties and nanostructuration. Thus, this work exhibits a method to induce and/or improve the mechanical antimicrobial behavior of surfaces via application of a magnetic field, as an alternative or in combination with chemical methods, which are losing effectiveness due to the increase of antibiotic resistance.FCT -Fundação para a Ciência e a Tecnologia(P2018/NMT-4321)info:eu-repo/semantics/publishedVersio

The malignant breast lesions characterization by use of the DW-magnetic resonance imaging

Objetivos – Com este estudo pretendeu-se i) avaliar o contributo da aplicação da sequência de difusão na caracterização das lesões mamárias malignas; ii) considerar se a sequência de difusão deve incorporar o protocolo standard em RM mamária e iii) correlacionar os resultados dos valores de coeficiente aparente de difusão (ADC) e os resultados histológicos. Metodologia – A amostra incluiu 18 pacientes do sexo feminino, com idades compreendidas entre 38 e 71 anos, que apresentavam lesões mamárias malignas confirmadas histologicamente. Foi adicionado ao protocolo de RM mamária a sequência de difusão, de modo a calcular os valores de ADC das lesões observadas. Resultados – Verificou-se que a range de valores de ADC para lesões malignas em ROI’s calculados no centro da lesão apresentavam uma média e desvio-padrão de (0,89 ± 0,14x10-3mm2/s). O método da utilização dos valores de ADC na caracterização de lesões mamárias malignas demonstrou uma sensibilidade de 100%. Conclusões – Neste estudo, com uma sensibilidade de 100%, a ponderação em difusão demonstrou ser uma técnica vantajosa na caracterização de lesões mamárias malignas pelo que se sugere a sua introdução no protocolo standard da RM mamária. ABSTRACT - Aims – The aim of this study was i) to evaluate the potential of the DWI sequence in the characterization of malignant breast lesions; ii) to verify if this sequence should incorporate the breast MRI protocol and iii) to correlate the apparent diffusion coefficients (ADC) values and histological results. Methodology – The sample includes 18 female patients between the ages of 38 and 71 years, who presented with malignant breast lesion confirmed by histology. The DWI sequence was added to the MRI standard protocol to calculate the ADC values. Results – In the results obtained we observed that the range of the ADC values calculated in the center of the malignant lesions, showed a mean and standard deviation of 0.89 ± 0.14 x10-3 mm2 / s. This method of using the ADC values for the detection of malignant lesions showed a sensitivity of 100%. Conclusion – The DWI technique proved to be a useful method in the characterization of malignant breast lesions, as it showed a sensitivity of 100%, so we suggest its inclusion in the Breast MR standard protocol

Piezo- and magnetoelectric polymers as biomaterials for novel tissue engineering strategies

Tissue engineering and regenerative medicine are increasingly taking advantage of active materials, allowing to provide specific clues to the cells. In particular, the use of electroactive polymers that deliver an electrical signal to the cells upon mechanical solicitation, open new scientific and technological opportunities, as they in fact mimic signals and effects that occur in living tissues, allowing the development of suitable microenvironments for tissue regeneration. Thus, a novel overall strategy for bone and muscle tissue engineering was developed based on the fact that these cells type are subjected to mechano-electrical stimuli in their in vivo microenvironment and that piezo- and magnetoelectric polymers, used as scaffolds, are suitable for delivering those cues. The processing and functional characterizations of piezoelectric and magnetoelectric polymers based on poly(vinylindene fluoride) and poly-L-lactic acid in a variety of shapes, from microspheres to electrospun mats and three dimensional scaffolds, are shown as well as their performance in the development of novel bone and muscle tissue engineering.The authors thank the Portuguese Fundação para a Ciência e Tecnologia (FCT) for financial support under Strategic Funding UID/FIS/04650/2013 and project PTDC/EEI-SII/5582/2014, including FEDER funds, UE. The authors also thank the FCT for financial support under grants SFRH/BPD/90870/2012 (CR), SFRH/BPD/121526/2016 (DMC), SFRH/BD/111478/2015 (SR) and SFRH/BPD/121464/2016 (MMF). Financial support from the Spanish Ministry of Economy and Competitiveness (MINECO) through the project MAT2016-76039-C4-3-R (AEI/FEDER, UE) (including the FEDER financial support) and from the Basque Government Industry Department under the ELKARTEK Program is also acknowledged.info:eu-repo/semantics/publishedVersio

Analysis of the coupling between a single-mode fiber to a multi-core fiber with long-period gratings

In this paper, the coupling of a single source injected in a single-mode fiber to all the cores of a multi-core fiber is theoretically studied. The power transfer between the core and the cladding of a fiber is promoted by long-period gratings. To promote the power transfer between the fibers, we considered cladding modes with similar effective refractive index. The results show that the coupling is possible, but the design still needs to be optimized to maximize the power transfer.publishe

Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells

The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. This work describes the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. Since initial cell density and aggregate size have an important impact in the expansion and commitment of these cells into a particular lineage, a combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 µm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was then performed in 50 mL spinner flasks, and process optimization using a factorial design approach was developed involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures, that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that, after replating, retained more than 60% of Pax6-positive neural cells. Furthermore, after scalable differentiation, hiPSC-derived neural progenitors still retained their multipotent potential, being able to give rise to neuronal and glial cells. The results presented in this work should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using totally controlled, automated and reproducible large-scale bioreactor culture systems

New textile for personal protective equipment—plasma chitosan/silver nanoparticles nylon fabric

Fabric structures are prone to contamination with microorganisms, as their morphology and ability to retain moisture creates a proper environment for their growth. In this work, a novel, easily processed and cheap coating for a nylon fabric with antimicrobial characteristics was devel- oped. After plasma treatment, made to render the fabric surface more reactive sites, the fabric was impregnated with chitosan and silver nanoparticles by simply dipping it into a mixture of different concentrations of both components. Silver nanoparticles were previously synthesized using the Lee–Meisel method, and their successful obtention was proven by UV–Vis, showing the presence of the surface plasmon resonance band at 410 nm. Nanoparticles with 25 nm average diameter observed by STEM were stable, mainly in the presence of chitosan, which acted as a surfactant for silver nanoparticles, avoiding their aggregation. The impregnated fabric possessed bactericidal activ- ity higher for Gram-positive Staphylococcus aureus than for Gram-negative Pseudomonas aeruginosa bacteria for all combinations. The percentage of live S. aureus and P. aeruginosa CFU was reduced to less than 20% and 60%, respectively, when exposed to each of the coating combinations. The effect was more pronounced when both chitosan and silver were present in the coating, suggesting an effective synergy between these components. After a washing process, the antimicrobial effect was highly reduced, suggesting that the coating is unstable after washing, being almost completely removed from the fabric. Nevertheless, the new-coated fabric can be successfully used in single-use face masks. To our knowledge, the coating of nylon fabrics intended for face-mask material with both agents has never been reported.This study was supported by the Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of UIDB/04469/2020 unit, and BioTecNorte operation (NORTE-01-0145-FEDER-000004). funded by the European Regional Development, Fund under the scope of Norte2020—Programa Operacional Regional do Norte