Genome-scale metabolic network reconstruction of Polaromonas sp. strain JS666: analysis of cDCE degradation rates and design of experiments for bioremediation improvement

Release of chloroethene compounds into the environment often results in groundwater contamination, which puts people at risk of exposure by drinking contaminated water. cDCE (cis-1,2-dichloroethene) accumulation on subsurface environments is a common environmental problem due to stagnation and partial degradation of other precursor chloroethene species. Polaromonas sp. strain JS666 apparently requires no exotic growth factors to be used as a bioaugmentation agent for aerobic cDCE degradation. Although being the only suitable microorganism found capable of such, further studies are needed for improving the intrinsic bioremediation rates and fully comprehend the metabolic processes involved. In order to do so, a metabolic model, iJS666, was reconstructed from genome annotation and available bibliographic data. FVA (Flux Variability Analysis) and FBA (Flux Balance Analysis) techniques were used to satisfactory validate the predictive capabilities of the iJS666 model. The iJS666 model was able to predict biomass growth for different previously tested conditions, allowed to design key experiments which should be done for further model improvement and, also, produced viable predictions for the use of biostimulant metabolites in the cDCE biodegradation.

Cellulose-based composites as functional conductive materials for printed electronics

In this work, cellulose-based electro and ionic conductive composites were developed for application in cellulose based printed electronics. Electroconductive inks were successfully formulated for screen-printing using carbon fibers (CFs) and multi-walled carbon nanotubes (MWCNTs) as conductive functional material and cellulose derivatives working as binder. The formulated inks were used to fabricate conductive flexible and disposable electrodes on paper-based substrates. Interesting results were obtained after 10 printing passes and drying at RT of the ink with 10 % wt. of pristine CFs and 3% wt. of carboxymethyl cellulose (CMC), exhibiting a resistivity of 1.03 Ωcm and a resolution of 400 μm. Also, a resistivity of 0.57 Ωcm was obtained for only one printing pass using an ink based on 0.5 % wt. MWCNTs and 3 % wt. CMC. It was also demonstrated that ionic conductive cellulose matrix hydrogel can be used in electrolyte-gated transistors (EGTs). The electrolytes revealed a double layer capacitance of 12.10 μFcm-2 and ionic conductivity of 3.56x10-7 Scm-1. EGTs with a planar configuration, using sputtered GIZO as semiconducting layer, reached an ON/OFF ratio of 3.47x105, a VON of 0.2 V and a charge carrier mobility of 2.32 cm2V-1s-1.

Usefulness of zebrafish model to assess glomerular and tubular alterations induced by tenofovir

Tenofovir (TFV) is one of the most used antiretroviral drugs. However, it is associated with tubular damage with mitochondria as a possible target. Tubulopathy precedes glomerular dysfunction, thus classic markers of renal function like the glomerular filtration rate (GFR) do not detect early TFV damage. Prediction and management of drug induced renal injury (DIRI) rely on the mechanisms of the drug insult and in optimal animal models to explore it. Zebrafish (Danio rerio) offers unique advantages for assessing DIRI, since the pronephros is structurally very similar to its human counterpart and is fully developed at 3.5 days postfertilization. The main aim of the present work was to evaluate the effects of TFV, as well as its pro-drug, tenofovir disoproxil fumarate (TDF), on the GFR and in mitochondria morphology in tubular cells of zebrafish larvae. Lethality curves were performed to understand the relationship between drug concentration and lethality. LC10 was selected to explore the renal function using the FITC-inulin assay and to analyze the mitochondrial toxicity by electron microscopy on larvae exposed to TDF, TFV, paracetamol and gentamicin (positive controls) or water (negative control). Lethality curves showed that gentamicin was the most lethal drug, followed by TDF, TFV and paracetamol. Gentamicin and paracetamol decreased the GFR, but no differences were found for either TDF or TFV, when compared to controls (%FITC Control = 33±8; %FITC TDF = 35±10; %FITC TFV = 30±10; %FITC Gentamicin = 46±17; %FITC Paracetamol = 83±14). Tubular mitochondria from treated larvae were notably different from non-treated larvae, showing swelling, irregular shapes, decreased mitochondria network, cristae disruption and loss of matrix granules. These results are in agreement with the effects of these drugs in humans and thus, demonstrate that zebrafish larvae can be a good model to assess the functional and structural damage associated with DIRI.

Cell functional enviromics: applications to Pichia pastoris

The present PhD thesis develops the cell functional enviromics (CFE) method to investigate the relationship between environment and cellular physiology. CFE may be defined as the envirome-wide cellular function reconstruction through the collection and systems-level analysis of dynamic envirome data. Throughout the thesis, CFE is illustrated by two main applications to cultures of a constitutive P. pastoris X33 strain expressing a scFv antibody fragment. The first application addresses the challenge of culture media development. A dataset was built from 26 shake flask experiments, with variations in trace elements concentrations and basal medium dilution based on the standard BSM+PTM1. Protein yield showed high sensitivity to culture medium variations, while biomass was essentially determined by BSM dilution. High scFv yield was associated with high overall metabolic fluxes through central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy-generating pathways. CFE identified three cellular functions (growth, energy generation and by-product formation) that together described 98.8% of the variance in observed fluxes. Analyses of how medium factors relate to identified cellular functions showed iron and manganese at concentrations close to PTM1 inhibit overall metabolic activity. The second application addresses bioreactor operation. Pilot 50 L fed-batch cultivations, followed by 1H-NMR exometabolite profiling, allowed the acquisition of data for 21 environmental factors over time. CFE identified five major metabolic pathway groups that are frequently activated by the environment. The resulting functional enviromics map may serve as template for future optimization of media composition and feeding strategies for Pichia pastoris. The present PhD thesis is a step forward towards establishing the foundations of CFE that is still at its infancy. The methods developed herein are a contribution for changing the culture media and process development paradigm towards a holistic and systematic discipline in the future.

Photochemical synthesis and functional transformations of bicyclic vinyl aziridines

Aziridines, a class of organic compounds containing a three membered heterocycle with a nitrogen atom, are extremely valuable molecules in organic and medicinal chemistry. They are frequently used as versatile precursors in the synthesis of natural products, and many biologically active molecules possess the aziridine moiety. The reactivity of aziridines has been studied, for example, in ring-opening reactions with thiols. However, not much interest seems to be given to reactions of aziridines in aqueous media, despite the numberless advantages of using water as solvent in organic chemistry. The nucleophilic ring-opening reaction of aziridines in aqueous media was here explored. Following the Kaplan aziridine synthetic methodology, in which pyridinium salts undergo a photochemical transformation to give bicyclic vinyl aziridines, new aziridines were synthetized. Their nucleophilic ring-opening reaction in water under physiological conditions was investigated and a range of sulphur, nitrogen, carbon and oxygen nucleophiles tested. Thiols, anilines and azide proved to be good nucleophiles to react with the aziridines, giving the ring-opening product in moderate to good yields. The best results were obtained with thiols, more specifically with cysteine-derived nucleophiles. Preliminary results show that these bicyclic vinyl aziridines can modify calcitonin, a peptide containing two cysteine amino acids residues, grating them the potential to be used in bioconjugation as ligands to cysteine-containing proteins, or even as enzyme inhibitors of, for example, cysteine proteases. Additionally, exploratory investigations suggest that the separation of both enantiomers of the bicyclic vinyl aziridine can be performed by taking advantage of an enzymatic methodology for the resolution of racemic secondary alcohols. Both enantiomers would be highly valuable as precursors in the synthesis of enantiomerically pure molecules, as no other method is currently reported for their separation.

Conflict in cross-functional teams

The present study is a qualitative analysis of the tensions that occur in cross-functional teams (CFTs) and the way in which they can affect the teams' accomplishments. The research is focused on a single case, COHTTEC 2005, a program aimed at transforming new technologies into businesses that took place in Lisbon, Portugal, starting March 2005. In this program, participants were organized in cross-functional teams that included researchers and management students. Semi-structured interviews were made to a sample of 17 participants. These interviews were recorded and transcribed. A table was then elaborated based on the primary data provided by the subjects and these data were classified in different categories. A three-stage model of group dynamics is proposed. The major findings of this project point to the role of interpersonal relationships on group outcomes. More specifically, several patterns associated with team malfunction were uncovered, the clearest ones being autocratic decision-making and arrogant behavior from some team members, as well as escalation in relationship conflict. Implications from these results are discussed, both for future research and for practice.