Volatile terpenoids and C13 norisoprenoids in wines: development of rapid methods of analysis and evaluation of the sesquiterpenoids biological potential

Vitis vinifera L., the most widely cultivated fruit crop in the world, was the starting point for the development of this PhD thesis. This subject was exploited following on two actual trends: i) the development of rapid, simple, and high sensitive methodologies with minimal sample handling; and ii) the valuation of natural products as a source of compounds with potential health benefits. The target group of compounds under study were the volatile terpenoids (mono and sesquiterpenoids) and C13 norisoprenoids, since they may present biological impact, either from the sensorial point of view, as regards to the wine aroma, or by the beneficial properties for the human health. Two novel methodologies for quantification of C13 norisoprenoids in wines were developed. The first methodology, a rapid method, was based on the headspace solid-phase microextraction combined with gas chromatography-quadrupole mass spectrometry operating at selected ion monitoring mode (HS-SPME/GC-qMS-SIM), using GC conditions that allowed obtaining a C13 norisoprenoid volatile signature. It does not require any pre-treatment of the sample, and the C13 norisoprenoid composition of the wine was evaluated based on the chromatographic profile and specific m/z fragments, without complete chromatographic separation of its components. The second methodology, used as reference method, was based on the HS-SPME/GC-qMS-SIM, allowing the GC conditions for an adequate chromatographic resolution of wine components. For quantification purposes, external calibration curves were constructed with β-ionone, with regression coefficient (r2) of 0.9968 (RSD 12.51 %) and 0.9940 (RSD of 1.08 %) for the rapid method and for the reference method, respectively. Low detection limits (1.57 and 1.10 μg L-1) were observed. These methodologies were applied to seventeen white and red table wines. Two vitispirane isomers (158-1529 L-1) and 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) (6.42-39.45 μg L-1) were quantified. The data obtained for vitispirane isomers and TDN using the two methods were highly correlated (r2 of 0.9756 and 0.9630, respectively). A rapid methodology for the establishment of the varietal volatile profile of Vitis vinifera L. cv. 'Fernão-Pires' (FP) white wines by headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (HS-SPME/GCxGC-TOFMS) was developed. Monovarietal wines from different harvests, Appellations, and producers were analysed. The study was focused on the volatiles that seem to be significant to the varietal character, such as mono and sesquiterpenic compounds, and C13 norisoprenoids. Two-dimensional chromatographic spaces containing the varietal compounds using the m/z fragments 93, 121, 161, 175 and 204 were established as follows: 1tR = 255-575 s, 2tR = 0,424-1,840 s, for monoterpenoids, 1tR = 555-685 s, 2tR = 0,528-0,856 s, for C13 norisoprenoids, and 1tR = 695-950 s, 2tR = 0,520-0,960 s, for sesquiterpenic compounds. For the three chemical groups under study, from a total of 170 compounds, 45 were determined in all wines, allowing defining the "varietal volatile profile" of FP wine. Among these compounds, 15 were detected for the first time in FP wines. This study proposes a HS-SPME/GCxGC-TOFMS based methodology combined with classification-reference sample to be used for rapid assessment of varietal volatile profile of wines. This approach is very useful to eliminate the majority of the non-terpenic and non-C13 norisoprenic compounds, allowing the definition of a two-dimensional chromatographic space containing these compounds, simplifying the data compared to the original data, and reducing the time of analysis. The presence of sesquiterpenic compounds in Vitis vinifera L. related products, to which are assigned several biological properties, prompted us to investigate the antioxidant, antiproliferative and hepatoprotective activities of some sesquiterpenic compounds. Firstly, the antiradical capacity of trans,trans-farnesol, cis-nerolidol, α-humulene and guaiazulene was evaluated using chemical (DPPH• and hydroxyl radicals) and biological (Caco-2 cells) models. Guaiazulene (IC50= 0.73 mM) was the sesquiterpene with higher scavenger capacity against DPPH•, while trans,trans-farnesol (IC50= 1.81 mM) and cis-nerolidol (IC50= 1.48 mM) were more active towards hydroxyl radicals. All compounds, with the exception of α-humulene, at non-cytotoxic levels (≤ 1 mM), were able to protect Caco-2 cells from oxidative stress induced by tert-butyl hydroperoxide. The activity of the compounds under study was also evaluated as antiproliferative agents. Guaiazulene and cis-nerolidol were able to more effectively arrest the cell cycle in the S-phase than trans,trans-farnesol and α-humulene, being the last almost inactive. The relative hepatoprotection effect of fifteen sesquiterpenic compounds, presenting different chemical structures and commonly found in plants and plant-derived foods and beverages, was assessed. Endogenous lipid peroxidation and induced lipid peroxidation with tert-butyl hydroperoxide were evaluated in liver homogenates from Wistar rats. With the exception of α-humulene, all the sesquiterpenic compounds under study (1 mM) were effective in reducing the malonaldehyde levels in both endogenous and induced lipid peroxidation up to 35% and 70%, respectively. The developed 3D-QSAR models, relating the hepatoprotection activity with molecular properties, showed good fit (R2LOO > 0.819) with good prediction power (Q2 > 0.950 and SDEP < 2%) for both models. A network of effects associated with structural and chemical features of sesquiterpenic compounds such as shape, branching, symmetry, and presence of electronegative fragments, can modulate the hepatoprotective activity observed for these compounds. In conclusion, this study allowed the development of rapid and in-depth methods for the assessment of varietal volatile compounds that might have a positive impact on sensorial and health attributes related to Vitis vinifera L. These approaches can be extended to the analysis of other related food matrices, including grapes and musts, among others. In addition, the results of in vitro assays open a perspective for the promising use of the sesquiterpenic compounds, with similar chemical structures such as those studied in the present work, as antioxidants, hepatoprotective and antiproliferative agents, which meets the current challenges related to diseases of modern civilization.

Development of infrared spectroscopy for assessing bacterial quality in foods

Rapid and specific detection of foodborne bacteria that can cause food spoilage or illness associated to its consumption is an increasingly important task in food industry. Bacterial detection, identification, and classification are generally performed using traditional methods based on biochemical or serological tests and the molecular methods based on DNA or RNA fingerprints. However, these methodologies are expensive, time consuming and laborious. Infrared spectroscopy is a reliable, rapid, and economic technique which could be explored as a tool for bacterial analysis in the food industry. In this thesis it was evaluated the potential of IR spectroscopy to study the bacterial quality of foods. In Chapter 2, it was developed a calibration model that successfully allowed to predict the bacterial concentration of naturally contaminated cooked ham samples kept at refrigeration temperature during 8 days. In this part, it was developed the methodology that allowed the best reproducibility of spectra from bacteria colonies with minimal sample preparation, which was used in the subsequent work. Several attempts trying different resolutions and number of scans in the IR were made. A spectral resolution of 4 cm-1, with 32 scans were the settings that allowed the best results. Subsequently, in Chapter 3, it was made an attempt to identify 22 different foodborne bacterial genera/species using IR spectroscopy coupled with multivariate analysis. The principal component analysis, used as an exploratory technique, allowed to form distinct groups, each one corresponding to a different genus, in most of the cases. Then, a hierarchical cluster analysis was performed to further analyse the group formation and the possibility of distinction between species of the same bacterial genus. It was observed that IR spectroscopy not only is suitable to the distinction of the different genera, but also to differentiate species of the same genus, with the simultaneous use of principal component analysis and cluster analysis techniques. The utilization of IR spectroscopy and multivariate statistical analysis were also investigated in Chapter 4, in order to confirm the presence of Listeria monocytogenes and Salmonella spp. isolated from contaminated foods, after growth in selective medium. This would allow to substitute the traditional biochemical and serological methods that are used to confirm these pathogens and that delay the obtainment of the results up to 2 days. The obtained results allowed the distinction of 3 different Listeria species and the distinction of Salmonella spp. from other bacteria that can be mistaken with them. Finally, in chapter 5, high pressure processing, an emerging methodology that permits to produce microbiologically safe foods and extend their shelf-life, was applied to 12 foodborne bacteria to determine their resistance and the effects of pressure in cells. A treatment of 300 MPa, during 15 minutes at room temperature was applied. Gram-negative bacteria were inactivated to undetectable levels and Gram-positive showed different resistances. Bacillus cereus and Staphylococcus aureus decreased only 2 logs and Listeria innocua decreased about 5 logs. IR spectroscopy was performed in bacterial colonies before and after HPP in order to investigate the alterations of the cellular compounds. It was found that high pressure alters bands assigned to some cellular components as proteins, lipids, oligopolysaccharides, phosphate groups from the cell wall and nucleic acids, suggesting disruption of the cell envelopes. In this work, bacterial quantification and classification, as well as assessment of cellular compounds modification with high pressure processing were successfully performed. Taking this into account, it was showed that IR spectroscopy is a very promising technique to analyse bacteria in a simple and inexpensive manner.

Development and Progress as Historical Phenomena in Tanzania: “Maendeleo? We Had That in the Past”

Academic discussions of development continue to grow, yet critical engagements with communities affected by development interventions remain limited. Drawing from life history interviews conducted in southern Tanzania, this article details the varied experiences of development interventions among older people and how these affect broader understandings of progress. Many juxtapose their negative views of ujamaa villagization with more positive recollections of previous interventions (especially the Groundnut Scheme), which are infused with what is described here as “development nostalgia.” Perceptions of the past clearly inform the social, political, and economic aspirations forwarded today, with the richness of the constructed narratives adding further nuance to existing depictions of Tanzanian historiography.

Detection of mineralized structures in very early stages of development of Marine Teleostei using a modified Alcian blue-Alizarin red double staining technique for bone and cartilage

We have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100-200 pm in length can be clearly visualized. The method is suitable for both ontogenic studies during early stages of skeletal development in most marine fishes (e.g., Sparus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 1000/0 ethanol for up to two years.

Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been developed, using different viral vectors (adenovirus and Sendai virus), transient plasmid transfection, mRNA transduction, protein transduction and use of small molecules. However, these methods suffer from low efficiencies; can be extremely labor intensive, or both. An additional method makes use of the piggybac transposon, which has the advantage of inserting its payload into the host genome and being perfectly excised upon re-expression of the transposon transposase. Briefly, a policistronic cassette expressing Oct4, Sox2, Klf4 and C-Myc flanked by piggybac terminal repeats is delivered to the cells along with a plasmid transiently expressing piggybac transposase. Once reprogramming occurs, the cells are re-transfected with transposase and subclones free of tranposon integrations screened for. The procedure is therefore very labor intensive, requiring multiple manipulations and successive rounds of cloning and screening. The original method for reprogramming with the the PiggyBac transposon was created by Woltjen et al in 2009 (schematized here) and describes a process with which it is possible to obtain insert-free iPSc. Insert-free iPSc enables the establishment of better cellular models of iPS and adds a new level of security to the use of these cells in regenerative medicine. Due to the fact that it was based on several low efficiency steps, the overall efficiency of the method is very low (<1%). Moreover, the stochastic transfection, integration, excision and the inexistence of an active way of selection leaves this method in need of extensive characterization and screening of the final clones. In this work we aime to develop a non-integrative iPSc derivation system in which integration and excision of the transgenes can be controlled by simple media manipulations, avoiding labor intensive and potentially mutagenic procedures. To reach our goal we developed a two vector system which is simultaneously delivered to original population of fibroblasts. The first vector, Remo I, carries the reprogramming cassette and GFP under the regulation of a constitutive promoter (CAG). The second vector, Eneas, carries the piggybac transposase associated with an estrogen receptor fragment (ERT2), regulated in a TET-OFF fashion, and its equivalent reverse trans-activator associated with a positive-negative selection cassette under a constitutive promoter. We tested its functionality in HEK 293T cells. The protocol is divided in two the following steps: 1) Obtaining acceptable transfection efficiency into human fibroblasts. 2) Testing the functionality of the construct 3) Determining the ideal concentration of DOX for repressing mPB-ERT2 expression 4) Determining the ideal concentration of TM for transposition into the genome 5) Determining the ideal Windows of no DOX/TM pulse for transposition into the genome 6) 3, 4 and 5) for transposition out of the genome 7) Determination of the ideal concentration of GCV for negative selection We successfully demonstrated that ENEAS behaved as expected in terms of DOX regulation of the expression of mPB-ERT2. We also demonstrated that by delivering the plasmid into 293T HEK cells and manipulating the levels of DOX and TM in the medium, we could obtain puromycin resistant lines. The number of puromycin resistant colonies obtained was significantly higher when DOX as absent, suggesting that the colonies resulted from transposition events. Presence of TM added an extra layer of regulation, albeit weaker. Our PCR analysis, while not a clean as would be desired, suggested that transposition was indeed occurring, although a background level of random integration could not be ruled out. Finally, our attempt to determine whether we could use GVC to select clones that had successfully mobilized PB out of the genome was unsuccessful. Unexpectedly, 293T HEK cells that had been transfected with ENEAS and selected for puromycin resistance were insensitive to GCV.

The Development of Environmental Assessment Tools to Support the Creation of Dementia Friendly Care Environments: Innovative Practice

The need for more dementia friendly design in hospitals and other care settings is now widely acknowledged. Working with 26 NHS Trusts in England as part of a Department of Health commissioned programme, The King’s Fund developed a set of overarching design principles and an environmental assessment tool for hospital wards in 2012. Following requests from other sectors, additional tools were developed for hospitals, care homes, health centres and housing with care. The tools have proven to be effective in both disseminating the principles of dementia friendly design and in enabling the case to be made for improvements that have a positive effect on patient outcomes and staff morale. This paper reports on the development,use and review of the environmental assessment tools, including further work that is now being taken forward by The Association for Dementia Studies, University of Worcester.

Development and characterization of novel vehicles for topical drug delivery

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016

Governing through community? A comparative study of changing management practices in mixed tenure housing development

An important feature of UK housing policy has been the promotion of consortia between local authorities, private developers and housing associations in order to develop mixed tenure estates to meet a wide range of housing needs. Central to this approach has been a focus on the management of neighbourhoods, based on the assumption that high densities and the inter-mixing of tenure exacerbates the potential for incivility and anti-social behaviour and exerts a disproportionate impact on residents' quality of life. Landlord strategies are therefore based on a need to address such issues at an early stage in the development. In some cases community-based, third sector organisations are established in order to manage community assets and to provide a community development service to residents. In others, a common response is to appoint caretakers and wardens to tackle social and environmental problems before they escalate and undermine residents’ quality of life. A number of innovative developments have promoted such neighbourhood governance approaches to housing practice by applying community development methods to address potential management problems. In the process, there is an increasing trend towards strategies that shape behaviour, govern ethical conduct, promote aesthetic standards and determine resident and landlord expectations. These processes can be related to the wider concept of governmentality whereby residents are encouraged to become actively engaged in managing their own environments, based on the assumption that this produces more cohesive, integrated communities and projects positive images. Evidence is emerging from a number of countries that increasingly integrated and mutually supportive roles and relationships between public, private and third sector agencies are transforming neighbourhood governance in similar ways. This paper will review the evidence for this trend towards community governance in mixed housing developments by drawing on a series of UK case studies prepared for two national agencies in 2007. It will review in particular the contractual arrangements with different tenures, identify codes and guidelines promoting 'good neighbour' behaviour and discuss the role of community development trusts and other neighbourhood organisations in providing facilities and services, designed to generate a well integrated community. The second part of the paper will review evidence from the USA and Australia to see how far there is a convergence in this respect in advanced economies. The paper will conclude by discussing the extent to which housing management practice is changing, particularly in areas of mixed development, whether there is a convergence in practice between different countries and how far these trends are supported by theories of governmentality.

How can economic and political liberalisation improve financial development in African countries?

The objective of this paper is to study the interactions between Economic liberalisation, Political liberalisation and Financial development in African countries. More specifically, we seek to establish the impact of Economic, Political and institutional openness on financial deepening. The empirical approach will be two-step procedure, first using a difference in difference method to show the various aspect of financial liberalisation on economic and political freedom while the second step will be using panel data techniques from period 1990 to 2005. The estimation results can be summarised as the following, first, Economic and financial liberalisation did account significantly for the financial development performance. While political stability show a positive overall effect on financial development, the association with Political freedom is consistent only after controlling the endogeneity of Political freedom on financial development. This result indicates that the transformation of the political and economic environment has improved the performance of the financial sector.

In-situ development of self-defensive antibacterial biomaterials: phenol-g-keratin-EC based bio-composites with characteristics for biomedical applications

Recently, the development of highly inspired biomaterials with multi-functional characteristics has gained considerable attention, especially in biomedical, and other health-related areas of the modern world. It is well-known that the lack of antibacterial potential has significantly limited biomaterials for many challenging applications such as infection free wound healing and/or tissue engineering etc. In this perspective, herein, a series of novel bio-composites with natural phenols as functional entities and keratin-EC as a base material were synthesised by laccase-assisted grafting. Subsequently, the resulting composites were removed from their respective casting surfaces, critically evaluated for their antibacterial and biocompatibility features and information is also given on their soil burial degradation profile. In-situ synthesised phenol-g-keratin-EC bio-composites possess strong anti-bacterial activity against Gram-positive and Gram-negative bacterial strains i.e., B. subtilis NCTC 3610, P. aeruginosa NCTC 10662, E. coli NTCT 10418 and S. aureus NCTC 6571. More specifically, 10HBA-g-keratin-EC and 20T-g-keratin-EC composites were 100% resistant to colonisation against all of the aforementioned bacterial strains, whereas, 15CA-g-keratin-EC and 15GA-g-keratin-EC showed almost negligible colonisation up to a variable extent. Moreover, at various phenolic concentrations used, the newly synthesised composites remained cytocompatible with human keratinocyte-like HaCaT, as an obvious cell ingrowth tendency was observed and indicated by the neutral red dye uptake assay. From the degradation point of view, an increase in the degradation rate was recorded during their soil burial analyses. Our investigations could encourage greater utilisation of natural materials to develop bio-composites with novel and sophisticated characteristics for potential applications.

Development of novel antibacterial active, HaCaT biocompatible and biodegradable CA-g-P(3HB)-EC biocomposites with caffeic acid as a functional entity

We have developed novel composites by grafting caffeic acid (CA) onto the P(3HB)-EC based material and laccase from Trametes versicolor was used for grafting purposes. The resulting composites were designated as CA-g-P(3HB)-EC i.e., P(3HB)-EC (control), 5CA-g-P(3HB)-EC, 10CA-g-P(3HB)-EC, 15CA-g-P(3HB)-EC and 20CA-g-P(3HB)-EC. An FT-IR (Fourier-transform infrared spectroscopy) was used to examine the functional and elemental groups of the control and laccase-assisted graft composites. Evidently, 15CA-g-P(3HB)-EC composite exhibited resilient antibacterial activity against Gram-positive and Gram-negative bacterial strains, respectively. Moreover, a significant level of biocompatibility and biodegradability of the CA-g-P(3HB)-EC composites was also achieved with the human keratinocytes-like HaCaT cells and soil burial evaluation, respectively. In conclusion, the newly developed novel composites with multi characteristics could well represent the new wave of biomaterials for medical applications, and more specifically have promising future in the infection free would dressings, burn and/or skin regeneration field due to their sophisticated characteristics.

A Communication Intervention for Training Southern European Oncologists to Recognize Psychosocial Morbidity in Cancer. I - Development of the Model and Preliminary Results on Physicians' Satisfaction

BACKGROUND: The detection of psychosocial distress is a significant communication problem in Southern Europe and other countries. Work in this area is hampered by a lack of data. Because not much is known about training aimed at improving the recognition of psychosocial disorders in cancer patients, we developed a basic course model for medical oncology professionals. METHODS: A specific educational and experiential model (12 hours divided into 2 modules) involving formal teaching (ie, journal articles, large-group presentations), practice in small groups (ie, small-group exercises and role playing), and discussion in large groups was developed with the aim of improving the ability of oncologists to detect emotional disturbances in cancer patients (ie, depression, anxiety, and adjustment disorders). RESULTS: A total of 30 oncologists from 3 Southern European countries (Italy, Portugal, and Spain) participated in the workshop. The training course was well accepted by most participants who expressed general satisfaction and a positive subjective perception of the utility of the course for clinical practice. Of the total participants, 28 physicians (93.3%) thought that had they been exposed to this material sooner, they would have incorporated the techniques received in the workshop into their practices; 2 participants stated they would likely have done so. Half of the doctors (n = 15) believed that their clinical communication techniques were improved by participating in the workshop, and the remaining half thought that their abilities to communicate with cancer patients had improved. CONCLUSIONS: This model is a feasible approach for oncologists and is easily applicable to various oncology settings. Further studies will demonstrate the effectiveness of this method for improving oncologists skills in recognizing emotional disorders in their patients with cancer.

Dissecting the function of the SpoIIIJ and YqjG membrane protein insertases during bacterial spore development

Dissertation presented to obtain the Ph.D degree in Biology, Microbial Biology

Notch1 and T-cell development: insights from conditional knockout mice.

Notch proteins influence cell-fate decisions in many developmental systems. Gain-of-function studies have suggested a crucial role for Notch1 signaling at several stages during lymphocyte development, including the B/T, alphabeta/gammadelta and CD4/CD8 lineage choices. Here, we critically re-evaluate these conclusions in the light of recent studies that describe inducible and tissue-specific targeting of the Notch1 gene.