Utilização de sensores de baixo custo na medição de monóxido de carbono no ar ambiente

Nos últimos anos tem-se assistido à introdução de novos dispositivos de medição da poluição do ar baseados na utilização de sensores de baixo custo. A utilização menos complexa destes sistemas, possibilita a obtenção de dados com elevada resolução temporal e espacial, abrindo novas oportunidades para diferentes metodologias de estudos de monitorização da poluição do ar. Apesar de apresentarem capacidades analíticas distantes dos métodos de referência, a utilização destes sensores tem sido sugerida e incentivada pela União Europeia no âmbito das medições indicativas previstas na Diretiva 2008/50/CE, com uma incerteza expandida máxima de 25%. O trabalho desenvolvido no âmbito da disciplina de Projeto consistiu na escolha, caracterização e utilização em medições reais de um sensor de qualidade do ar, integrado num equipamento protótipo desenvolvido com esse fim, visando obtenção uma estimativa da incerteza de medição associada à utilização deste dispositivo através da aplicação da metodologia de demonstração de equivalência de métodos de medição de qualidade do ar definida pela União Europeia. A pesquisa bibliográfica realizada permitiu constatar que o monóxido de carbono é neste momento o parâmetro de qualidade do ar que permite ser medido de forma mais exata através da utilização de sensores, nomeadamente o sensor eletroquímico da marca Alphasense, modelo COB4, amplamente utilizado em projetos de desenvolvimento neste cotexto de monitorização ambiental. O sensor foi integrado num sistema de medição com o objetivo de poder ser utlizado em condições de autonomia de fornecimento de energia elétrica, aquisição interna dos dados, tendo em consideração ser o mais pequeno possível e de baixo custo. Foi utlizado um sistema baseado na placa Arduino Uno com gravação de dados em cartão de memória SD, baterias e painel solar, permitindo para além do registo das tensões elétricas do sensor, a obtenção dos valores de temperatura, humidade relativa e pressão atmosférica, com um custo global a rondar os 300 euros. Numa primeira fase foram executados um conjunto de testes laboratoriais que permitiram a determinação de várias características de desempenho em dois sensores iguais: tempo de resposta, a equação modelo do sensor, avaliação da repetibilidade, desvio de curto e longo termo, interferência da temperatura e histerese. Os resultados demonstraram um comportamento dos sensores muito linear, com um tempo de resposta inferior a um minuto e com uma equação modelo do sensor dependente da variação da temperatura. A estimativa da incerteza expandida laboratorial ficou, para ambos os sensores, abaixo dos 10%. Após a realização de duas campanhas reais de medição de CO em que os valores foram muito baixos, foi realizada uma campanha de quinze dias num parque de estacionamento subterrâneo que permitiu a obtenção de concentrações suficientemente elevadas e a comparação dos resultados dos sensores com o método de referência em toda a gama de medição (0 a 12 mol.mol-1). Os valores de concentração obtidos pelos dois sensores demonstraram uma excelente correlação com o método de referência (r2≥0,998), obtendo-se resultados para a estimativa da incerteza expandida de campo inferiores aos obtidos para a incerteza laboratorial, cumprindo o objetivo de qualidade de dados definido para as medições indicativas de incerteza expandida máxima de 25%. Os resultados observados durante o trabalho realizado permitiram confirmar o bom desempenho que este tipo de sensor pode ter no âmbito de medições de poluição do ar com um caracter mais indicativo.

Sialyl Tn-Expressing Bladder Cancer Cells Induce a Tolerogenic Phenotype in Innate and Adaptive Immune Cells

Despite the wide acceptance that glycans are centrally implicated in immunity, exactly how they contribute to the tilt immune response remains poorly defined. In this study, we sought to evaluate the impact of the malignant phenotype-associated glycan, sialyl-Tn (STn) in the function of the key orchestrators of the immune response, the dendritic cells (DCs). In high grade bladder cancer tissue, the STn antigen is significantly overexpressed and correlated with the increased expression of ST6GALNAC1 sialyltransferase. Bladder cancer tissue presenting elevated expression of ST6GALNAC1 showed a correlation with increased expression of CD1a, a marker for bladder immature DCs and showed concomitant low levels of Th1-inducing cytokines IL-12 and TNF-α. In vitro, human DCs co-incubated with STn+ bladder cancer cells, had an immature phenotype (MHC-IIlow, CD80low and CD86low) and were unresponsive to further maturation stimuli. When contacting with STn+ cancer cells, DCs expressed significantly less IL-12 and TNF-α. Consistent with a tolerogenic DC profile, T cells that were primed by DCs pulsed with antigens derived from STn+ cancer cells were not activated and showed a FoxP3high IFN-γlow phenotype. Blockade of STn antigens and of STn+ glycoprotein, CD44 and MUC1, in STn+ cancer cells was able to lower the induction of tolerance and DCs become more mature. Overall, our data suggest that STn-expressing cancer cells impair DC maturation and endow DCs with a tolerogenic function, limiting their capacity to trigger protective anti-tumour T cell responses. STn antigens and, in particular, STn+ glycoproteins are potential targets for circumventing tumour-induced tolerogenic mechanisms.

Cerebrospinal Fluid Synaptic Proteins as Useful Biomarkers in Tyrosine Hydroxylase Deficiency

Tyrosine hydroxylase (TH) deficiency is an inborn error of dopamine biosynthesis and a cause of early parkinsonism. Two clinical phenotypes have been described. Type “B”: early onset severe encephalopathy; type “A”: later onset, less severe and better response to L-dopa. We aimed to study the expression of several key dopaminergic and gabaergic synaptic proteins in the cerebrospinal fluid (CSF) of a series of patients with TH deficiency and their possible relation with the clinical phenotype and response to L-DOPA. Dopamine transporter (DAT), D2-receptor and vesicularmonoamine transporter (VMAT2)weremeasured in the CSF of 10 subjectswith THdeficiency byWestern blot analysis. In 3 patients, data of pre- and post-treatmentwith L-DOPA were available, and in one of them, GABA vesicular transporter was determined. Results were compared to an age-matched control population. The concentration of D2-receptors in CSFwas significantly higher in patients with TH deficiency than in controls. Similarly, DAT and vesicular monoamine transporter type 2 were up-regulated. Studies performed before LDOPA, and on L-DOPA therapy showed a paradoxical response with D2 receptor expression increase as L-Dopa doses and homovanillic concentration gradually raised in a B phenotype patient. The opposite results were found in two patients with A phenotype. However, this is a very small sample, and further studies are needed to conclude robust differences between phenotypes. Synaptic proteins are detectable in the CSF and their quantification can be useful for understanding the pathophysiology of neurotransmitter defects and potentially to adjust and personalize treatments in the future.

Gene Expression Profiling and Association Studies Implicate the Neuregulin Signaling Pathway in Behçet's Disease Susceptibility

Behçet's disease (BD) is a complex disease with genetic and environmental risk factors implicated in its etiology; however, its pathophysiology is poorly understood. To decipher BD's genetic underpinnings, we combined gene expression profiling with pathway analysis and association studies. We compared the gene expression profiles in peripheral blood mononuclear cells (PBMCs) of 15 patients and 14 matched controls using Affymetrix microarrays and found that the neuregulin signaling pathway was over-represented among the differentially expressed genes. The Epiregulin (EREG), Amphiregulin (AREG), and Neuregulin-1 (NRG1) genes of this pathway stand out as they are also among the top differentially expressed genes. Twelve haplotype tagging SNPs at the EREG-AREG locus and 15 SNPs in NRG1 found associated in at least one published BD genome-wide association study were tested for association with BD in a dataset of 976 Iranian patients and 839 controls. We found a novel association with BD for the rs6845297 SNP located downstream of EREG, and replicated three associations at NRG1 (rs4489285, rs383632, and rs1462891). Multifactor dimensionality reduction analysis indicated the existence of epistatic interactions between EREG and NRG1 variants. EREG-AREG and NRG1, which are members of the epidermal growth factor (EGF) family, seem to modulate BD susceptibility through main effects and gene–gene interactions. These association findings support a role for the EGF/ErbB signaling pathway inBD pathogenesis that warrants further investigation and highlight the importance of combining genetic and genomic approaches to dissect the genetic architecture of complex diseases.

Food waste valorization through the production of polyhydroxyalkanoates by mixed microbial cultures

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Exopolysaccharide production by Enterobacter A47: optimization of cultivation conditions and study of polymer functional properties

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Quantification and characterization of the phenolic compounds of kenaf leaves

Dissertation for the Master Degree in Technology and Food Security

Purification of complex biopharmaceuticals with new processes, advanced analytics and computer-aided process design tools

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Electrochemical and quantum chemical investigations of the insecticide fipronil.

This work describes an electrochemical and quantum chemical investigation of the fipronil insecticide. Cyclic voltammetry (CV) and square wave voltammetry (SWV) experiments were performed over a graphite-polyurethane (GPU) composite electrode. The fipronil molecule presents an one?electron irreversible oxidation reaction. Profiting the SWV signal a square wave stripping voltammetry (SWSV) procedure to determine the fipronil molecule in a 0.10 mol L-1 Britton-Robinson buffer solution, pH 8.0 was developed with accumulation potential and time of 0.50 V and 120 s, respectively. The limits of detection and quantification were 0.80 and 2.67 ?g L-1, respectively. Recovery tests were performed in three natural waters samples with values ranging from 99.67 to 101.37%. Quantum chemical studies showed that the nitrogen atom of the pyrazole group is the most probable oxidation site of the fipronil molecule.

Design and characterization of novel biopolymeric structure using biocompatible ionic liquids

The main objective of this thesis was the development of polymeric structures from the dissolution of FucoPol, a bacterial exopolysaccharide (EPS), in a biocompatible ionic liquid, choline acetate. The FucoPol was produced by the bacteria Enterobacter A47 using glycerol as carbon source at controlled temperature and pH (30ºC and 7, respectively). At the end of 3 days it was produced 7 g/L of FucoPol. The net yield of Fucopol in glycerol (YP/S) was 0.22 g/g and the maximum productivity 2.37 g/L.d This polymer was characterized about its composition in sugars and acyl groups (by High-Performance Liquid Chromatography - HPLC), containing fucose (35 % mol), galactose (21 % mol), glucose (29 % mol), rhamnose (3% mol) and glucuronic acid (12% mol) as well as acetate (14.28 % mol), pyruvate (2.15 % mol) and succinate (1.80 % mol). Its content of water and ash was 15% p/p and 2% p/p, respectively, and the chemical bonds (determined by Infrared Spectroscopy - FT-IR) are consistent to the literature reports. However, due to limitations in Differential Scanning Calorimetry (DSC) equipment it was not possible to determine the glass transition temperature. In turn, the ionic liquid showed the typical behavior of a Newtonian fluid, glass transition temperature (determined by DSC) -98.03ºC and density 1.1031 g/cm3. The study of chemical bonds by FT-IR showed that amount of water (8.80%) influenced the visualization of the bands predicted to in view of their chemical structure. After the dissolution of the FucoPol in the ionic liquid at different temperatures (50, 60, 80 and 100 ° C) it was promoted the removal of this by the phase inversion method using deionized water as a solvent, followed by drying in an oven at 70 ° C. The mixtures before and after the phase inversion method were characterized through the studies mentioned above. In order to explore possible application field’s biocompatibility assays and collage on balsa wood tests were performed. It was found that the process of washing with water by the phase inversion method was not totally effective in removing the biocompatible ionic liquid, since all FucoPol – IL mixtures still contained ionic liquid in their composition as can be seen by the DSC results and FT-IR. In addition, washing the mixtures with water significantly altered the composition of FucoPol. However, these mixtures, that developed a viscous behavior typical of a non-Newtonian fluid (shear-thinning), have the potential to be applied in the biomedical field as well as biological glues.

Production and characterization of Chitin-Glucan Complex by Komagataella pastoris

This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)

Estudo da partição de fitase produzida por Aspergillus niger var. phoenicis utilizando bioconversão extrativa em sistemas de duas fases aquosas PEG/citrato

Existem diversos métodos tradicionais que são utilizados para extratir biomoléculas produzidas por fermentação convencional. Um método alternativo é o sistema de duas fases aquosas, o qual foi desenvolvido para a extração de bioprodutos. A bioconversão extrativa trata-se de um sistema de duas fases aquosas que integra cultivo microbiológico à produção e recuperação do bioproduto. Fitases são fosfatases específicas que estão envolvidas na catálise do ácido fitico. O objetivo deste trabalho foi estudar a partição da fitase produzida por A. niger var. phoenicis por bioconversão extrativa utilizando PEG/citrato. Realizou-se um planejamento fatorial completo 25, estudando as seguintes variáveis: massa molar do PEG, concentração do PEG, concentração de citrato, pH e agitação, onde obteve-se como variável-resposta o coeficiente de partição em atividade (KATIV). Neste trabalho conseguiu-se um coeficiente de partição de 25,77 utilizando MPEG (8000 g/mol), CPEG, (26,0% m/m), CCIT (20,0% m/m), pH (6,0) e agitação (100 rpm). Através dos resultados obtivos, pode-se concluir que a fitase utilizada no presente estudo apresenta uma tendência de particionar para a fase superior do sistema (K>1). A técnica de fermentação extrativa utilizando SDFA PEG/citrato demonstrou ser promissora para extração de fitase produzida por A. niger var. phoenicis, podendo ser aplicada na composição de rações comerciais.

Sistemas aquosos bifásicos uma ferramenta sustentável para a extração de ácido clavulânico a partir de diferentes fontes

O ácido clavulânico (AC) é um inibidor de β-lactamases que tem vindo a ser largamente utilizado na área médica. Embora seja de extrema importância, o desenvolvimento de processos alternativos de produção e purificação é ainda insignificante, sendo fundamental o estudo de técnicas de extração mais biocompatíveis, como os Sistemas Aquosos Bifásicos (SABs). Assim, este trabalho objetivou o estudo de Sistemas Aquosos Bifásicos baseados em polímeros como uma ferramenta alternativa para a extração de AC. Foram testados dois SPAB compostos por Polietileno Glicol (PEG) com massa molecular (M) de 4000 g/mol e Poliacrilato de Sódio de 8000 g/mol, nos quais foi alterado o eletrólito indutor da formação de fases, em particular, sulfato de sódio (Na2SO4,) e cloreto de sódio (NaCl). Ademais, este trabalho visou também avaliar a eficiência de extração do AC, bem como compreender o efeito dos contaminantes no processo de migração. Para tal, foi avaliada a extração do AC a partir de três fontes distintas: solução pura (99,9%); solução comercial (60%); diretamente a partir do sobrenadante de um meio fermentando de Streptomyces clavuligerus. Os resultados obtidos demonstraram que independentemente da fonte inicial do AC, ambos os SABs poliméricos promoveram uma partição preferencial do AC para a fase rica em PEG, sendo o coeficiente de partição maior nos sistemas com Na2SO4 do que com NaCl. Após identificar a grande capacidade de partição de AC, o SAB com PEG/NaPA/Na2SO4 foi também utilizado para avaliar a partição de proteínas presente no meio fermentado, sendo também obtida uma preferencial partição destas para a fase rica em PEG. Assim, apesar da baixa capacidade de purificação de AC frente a proteínas contaminantes, os SABs estudados demonstraram que podem ser uma técnica alternativa sustentável e bastante econômica para uma etapa inicial de clarificação/concentração de bioprodutos a partir de caldos fermentados.

Effects of residues on the degradation of PHA produced from mixed microbial cultures and processed in extrusion

Thermal degradation upon melting is one of the major drawbacks reported for polyhydroxyalkanoates (PHA). However, the role of residues originating from the fermentation and the extraction steps on the thermal stability of this class of biopolymers still needs to be clarified. In the particular case of PHA produced from mixed microbial cultures (MMC), this topic is even less documented in the literature. Here, two polyhydroxy(butyrate-co-valerate) (PHBV) produced from MMC enriched in PHA accumulating organisms and fed with cheese whey were studied. A micro extrusion line is used to produce filaments and assess the processability and the degradation of processed PHBV. The prototype micro extrusion line allows for studying grams of materials. The two PHBV contain 18 mol% HV. PHBV was recovered with 11 wt% residues, and further submitted to a purification procedure resulting in a second biopolyester containing less than 2 wt% impurities. The thermorheological characterization of the two PHBV is first presented, together with their semicrystalline properties. Then the processing windows of the two biopolyesters are presented. Finally, the properties of extruded filaments are reported and the thermomechanical degradation of PHBV is extensively studied. The structure was assessed by wide angle X-ray diffraction, mechanical and rheological properties are reported, thermal properties are studied with differential scanning calorimetry and thermogravimetric analysis, whereas Fourier Transform Infrared spectroscopy was used to assess the impact of the extrusion on PHBV chemical structure. All results obtained with the two PHBV are compared to assess the effects of residues on both PHBV processability and degradation.

Leaf micronutrient concentrations and potential photosynthesis in Ochroma pyramidale established in a degraded land

In the Brazilian Amazon, large areas of abandoned lands may revert to secondary forest. In the process, pioneer tree species have an important role to restore productivity in old fields and improve environmental conditions. To determine potential photosynthesis (Apot), stomatal conductance (g), transpiration (E), and leaf micronutrient concentrations in Ochroma pyramidale (Cav. ex Lam.) Urban a study was carried out in the Brazilian Amazon (01o 51' S; 60o 04' W). Photosynthetic parameters were measured at increasing [CO2], saturating light intensity (1 mmol (photons) m-2 s-1), and ambient temperature. The rate of electron-transport (J), Apot,and water-use efficiency (WUE) increased consistently at increasing internal CO2 concentration (Ci). Conversely, increasing [CO2] decreased gs, E, and photorespiration (Pr). At the CO2-saturated region of the CO2 response curve (1.1 mmol (CO2) mol-1(air), J was 120 μmol (e-) m-2s-1 and Apot reached up to 24 μmol (CO2) m-2s-1. Likewise, at saturating C1 g and E were 30 and 1.4 mmol (H2O) m-2s-1, respectively, and P 2 r about 1.5 μmol (CO2) m-2s-1. Foliar nutrients were 185, 134, 50, and 10 μmol (element) m-2 (leaf area) for Fe, Mn, Zn, and Cu, respectively. It was concluded that [CO ] probably limits light saturated photosynthesis in this site. Furthermore, from a nutritional point of view, the low Fe to Cu ratio (15:1) may reflect nutritional imbalance in O. pyramidale at this site.