Aspectos da interacção entre Phytophthora cinnamomi e a doença do declínio em Q. suber e Q. rotundifolia

Faculdade de Engenharia de Recursos Naturais, Univ. do Algarve, 2001

An expert model approach to assess the potential of non-wood forest products for forest owners

Mestrado em Engenharia Florestal e dos Recursos Naturais - Instituto Superior de Agronomia - UL

A viabilidade económica da gestão sustentável do montado de sobreiro. Caso de estudo - Aldeia das Amoreiras

Dissertação de Mestrado, Economia do Turismo e Desenvolvimento Regional, Faculdade de Economia, Universidade do Algarve, 2016

An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.

Participation of portuguese manufacturing industry in global value chains

Portugal’s manufacturing sector has a significant importance both in national income and employment. As has been pointed out by several researchers, the traditional methods of analysis fail to grasp all the dimensions of economic competitiveness. This dissertation is then, at its core, an analysis of Portugal’s manufacturing industry in terms of the latter’s value added to production and impact to employment under the framework of global value chains. The current dissertation seeks to study in which way the Portuguese manufacturing industry, and its respective sectors, has a direct and indirect impact on the creation of value added and employment and how this impact can be measured. For development of this work the input-output approach for calculation of multipliers and the new framework proposed by Timmer et al. (2013) for calculation of GVC income and GVC jobs indicators were used, elaborated on the basis of the WIOD project dataset. Moreover, to illustrate the application of the provided methodology the Portuguese textile industry was used as an example. It was found that the changes in final demand of such sectors as Pulp, Paper, Printing and Publishing; Machinery, Nec and Textiles and Textile Products would have a larger impact on generated value added than other manufacturing sectors. At the same time, employment created by the changes in final demand would be more impacted by such sectors as Food, Beverages and Tobacco; Wood and Products of Wood and Cork and Textiles and Textile Products. In this regard, the number of low-skilled workers in Portugal seems to be more effected by changes in final demand, than those occupied by higher -skilled individuals. Moreover, it was found that the distribution of GVC income and GVC jobs for the Portuguese manufacturing industry shares a similar outlook. However, upon closer inspection of GVC labour distribution by skill levels there seems to exist a general progression in which low-skilled jobs requirements are met by local resources, while the need for higher skilled jobs require a greater “off-shoring” of work The results obtained through calculations of presented multipliers provide a powerful tool for policy makers in strategic planning of development of national economy. Using the provided methodology and obtained results, a government and supranational organizations could define which industry would have the greatest impact for an additional unit of output generated through the economy, and thus define the sectors for further investments.

Production of bioactive metabolites by intestinal bacteria

The adult intestinal microbiota comprises a microbial ecosystem of approximately 100 trillion microorganisms, with specific bacterial communities holding distinct metabolic capabilities. Bacteria produce a range of bioactive compounds to survive unfavourable stimuli and to interact with other organisms, and generate several bioactive products during degradation of dietary constituents the host is not capable of digesting. This thesis addressed the impact of feeding potential probiotic bacteria and other dietary strategies such as pure fatty acids and prebiotics, on gut microbiota composition, short chain fatty acid (SCFA) production and modulation of metabolism in animal models. In the first experimental chapter (Chapter 2) a gas chromatography method for the quantification of SCFA was optimized and applied in the analysis of caecal samples obtained in animal studies described in other chapters of this thesis. In Chapter 3, t10, c12 CLA supplementation was shown to significantly alter murine gut microbiota composition and SCFA production rather than no supplementation. These changes were suggested to be extra factors affecting host lipid metabolism. Chapter 4 described the contrasting effects of CLA-producing strains, Bifidobacterium breve DPC 6330 and B. breve NCIMB 702258, on murine fat distribution/composition and gut microbiota composition, suggesting that these changes were most likely strain-dependent. In Chapter 5, dietary GABA-producing strain Lactobacillus brevis DPC 6108 was shown to significantly increase (p<0.05) serum insulin in healthy rats, leading to a second experiment using a type 1 diabetes rat model. Lb. brevis DPC 6108 administration did not change insulin levels in diabetic rats, but attenuated high levels of glucose when compared to diabetic control. However, an auto-immune-induced diabetes model was suggested as a better model to study GABA-related effects on diabetes. In Chapter 6 bovine milk oligosaccharides, 6’sialyllactose and Beneo Orafti P95 oligofructose supplementations were associated with depletion or reduction of less favourable bacteria, demonstrating that ingestion of these oligosaccharides might be a safe and effective approach to modulate populations of the intestinal microbiota. In Chapter 7 (General discussion) the major findings of all studies were reviewed and discussed.

Antibiotic resistance in the gut microbiota

Antibiotic resistance is an increasing threat to our ability to treat infectious diseases. Thus, understanding the effects of antibiotics on the gut microbiota, as well as the potential for such populations to act as a reservoir for resistance genes, is imperative. This thesis set out to investigate the gut microbiota of antibiotic treated infants compared to untreated controls using high-throughput DNA sequencing. The results demonstrated the significant effects of antibiotic treatment, resulting in increased proportions of Proteobacteria and decreased proportions of Bifidobacterium. The species diversity of bifidobacteria was also reduced. This thesis also highlights the ability of the human gut microbiota to act as an antibiotic resistance reservoir. Using metagenomic DNA extracted from faecal samples from adult males, PCR was employed to demonstrate the prevalence and diversity of aminoglycoside and β-lactam resistance genes in the adult gut microbiota and highlighted the merits of the approach adopted. Using infant faecal samples, we constructed and screened a second fosmid metagenomic bank for the same families of resistance genes and demonstrated that the infant gut microbiota is also a reservoir for resistance genes. Using in silico analysis we highlighted the existence of putative aminoglycoside and β-lactam resistance determinants within the genomes of Bifidobacterium species. In the case of the β- lactamases, these appear to be mis-annotated. However, through homologous recombination-mediated insertional inactivation, we have demonstrated that the putative aminoglycoside resistance proteins do contribute to resistance. In additional studies, we investigated the effects of short bowel syndrome on infant gut microbiota, the immune system and bile acid metabolism. We also sequenced the microbiota of the human vermiform appendix, highlighting its complexity. Finally, this thesis demonstrated the strain specific nature of 2 different probiotic CLA-producing Bifidobacterium breve on the murine gut microbiota.

The exploration of potato-associated bacteria in the Central Andean Highlands and their application in integrated crop management systems

Potato is the most important food crop after wheat and rice. A changing climate, coupled with a heightened consumer awareness of how food is produced and legislative changes governing the usage of agrochemicals, means that alternative more integrated and sustainable approaches are needed for crop management practices. Bioprospecting in the Central Andean Highlands resulted in the isolation and in vitro screening of 600 bacterial isolates. The best performing isolates, under in vitro conditions, were field trialled in their home countries. Six of the isolates, Pseudomonas sp. R41805 (Bolivia), Pseudomonas palleroniana R43631 (Peru), Bacillus sp. R47065, R47131, Paenibacillus sp. B3a R49541, and Bacillus simplex M3-4 R49538 (Ecuador), showed significant increase in the yield of potato. Using – omic technologies (i.e. volatilomic, transcriptomic, proteomic and metabolomic), the influence of microbial isolates on plant defence responses was determined. Volatile organic compounds of bacterial isolates were identified using GC/MS. RT-qPCR analysis revealed the significant expression of Ethylene Response Factor 3 (ERF3) and the results of this study suggest that the dual inoculation of potato with Pseudomonas sp. R41805 and Rhizophagus irregularis MUCL 41833 may play a part in the activation of plant defence system via ERF3. The proteomic analysis by 2-DE study has shown that priming by Pseudomonas sp. R41805 can induce the expression of proteins related to photosynthesis and protein folding in in vitro potato plantlets. The metabolomics study has shown that the total glycoalkaloid (TGA) content of greenhouse-grown potato tubers following inoculation with Pseudomonas sp. R41805 did not exceed the acceptable safety limit (200 mg kg-1 FW). As a result of this study, a number of bacteria have been identified with commercial potential that may offer sustainable alternatives in both Andean and European agricultural settings.

Life history of a native emydid turtle (Malaclemys terrapin centrata) on the remote oceanic islands of Bermuda

Diamondback terrapins (Malaclemys terrapin) are native to the remote oceanic islands of Bermuda and presently inhabit only four small brackish water ponds on a private golf course. The life history of this species is poorly understood on Bermuda and so the aim of this study was to fill these knowledge gaps, to compare the results with what is known from other areas in the North American range, and to inform the development of a local management plan. The results of a mark-recapture census revealed that ca. 100 individuals ≥81 mm straight carapace length live on Bermuda, of which nearly half (48.5%) were considered sexually mature. The population is dominated by females (sex ratio 2.9:1) and annual recruitment over the three year period was found to be extremely low (approximately two terrapins). Female diamondback terrapins in Bermuda nest almost exclusively within a limited number of sand bunkers on the golf course. Nesting commenced in late March or early April and ended in late August. Peak oviposition was observed in May and June. Clutch size averaged 5.1 eggs (range 0-10; SD 2.4) and the incubation period averaged 61.8 days (range 49-83; SD 10.5). Delayed emergence was documented, with 43.8% of the hatchlings remaining in their natal nests over the winter months. The mean annual hatching success rate was determined to be 19% (range 17.6-21; SD 1.9). Radio-telemetry was used to investigate the movements and survivorship of postemergent hatchling diamondback terrapins. The results indicated that mangrove swamps and grass-dominated marshes adjacent to the ponds are important developmental habitats for hatchlings. Yellow-crowned night herons (Nyctanassa violacea) were found to be significant predators of small terrapins during spring emergence. Small aquatic gastropods comprised 66.7% of the faecal samples analysed from the Bermudian population. Scavenged fish and vertebrate animal remains, terrestrial arthropods, polychaete worms and bivalves were consumed in lesser amounts. Sediment from the pond environment was found in 74% of the faecal samples analysed and is believed to have been incidentally ingested while foraging for the small benthic gastropods. Eco-toxicological analyses of the pond sediment, prey and terrapin eggs showed that the Bermudian diamondback terrapins live and feed in wetland habitats characterised by chronic, multifactorial contamination; principally total petroleum hydrocarbons, polycyclic aromatic hydrocarbons and a variety of heavy metals. This study found that some of those contaminants are accumulating in the gastropod prey as well as being transferred to terrapin eggs. This may be reducing the incidence of successful embryonic development for this species in Bermuda and may likely contribute to the observed low hatching rates. These collective findings indicate that the Bermudian population is very vulnerable to local extirpation.

From field to fermentation: characterisation and application of non-dairy cultures in dairy foods

This thesis investigates the phenotypic and genotypic diversity of non-dairy L. lactis strains and their application to dairy fermentations. A bank of non-dairy lactococci were isolated from grass, vegetables and the bovine rumen. Subsequent analysis of these L. lactis strains revealed seven strains to possess cremoris genotypes which did not correlate with their observed phenotypes. Multi-locus sequence typing (MLST) and average nucleotide identity (ANI) highlighted the genetic diversity of lactis and cremoris subspecies. The application of these non-dairy lactococci to cheese production was also assessed. In milk, non-dairy strains formed diverse volatile profiles and selected strains were used as adjuncts in a mini Gouda-type cheese system. Sensory analysis showed non-dairy strains to be strongly associated with the development of off-flavours and bitterness. However, microfluidisation appeared to reduce bitterness. A novel bacteriophage, ɸL47, was isolated using the grass isolate L. lactis ssp. cremoris DPC6860 as a host. The phage, a member of the Siphoviridae, possessed a long tail fiber, previously unseen in dairy lactococcal phages. Genome sequencing revealed ɸL47 to be the largest sequenced lactococcal phage to date and owing to the high % similarity with ɸ949, a second member of the 949 group. Finally, to identify and characterise specific genes which may be important in niche adaptation and for applications to dairy fermentations, comparative genome sequence analysis was performed on L. lactis from corn (DPC6853), the bovine rumen (DPC6853) and grass (DPC6860). This study highlights the contribution of niche specialisation to the intra-species diversity of L. lactis and the adaptation of this organism to different environments. In summary this thesis describes the genetic diversity of L. lactis strains from outside the dairy environment and their potential application in dairy fermentations.

Microbially derived bioactive peptides to improve human health

This thesis describes a study of various methods to produce bioactive peptides. Initially, the generation of anti-Cronobacter spp. peptides by fermentation of milk protein is described. Lactobacillus johnsonii DPC6026 was used to generate two previously described antimicrobial peptides. Phenotypic analysis indicated unsatisfactory casein hydrolysis. The genome of the strain was sequenced and annotated. Results showed a number of unique features present, most notably a large symmetrical inversion of approximately 750kb in comparison with the human isolate L. johnsonii NCC 533. The data suggest significant genetic diversity and intra-species genomic rearrangements within the L. johnsonii spp.. Cronobacter spp. have emerged as pathogens of concern to the powdered infant formula industry. Chapters 3 and 4 of this thesis describe novel methods to generate two antimicrobial peptides, Caseicin A and B. In Chapter 3 a bank of Bacillus strains was generated and investigated for caseicin production. Following casein hydrolysis by specific B. cereus and B. thuringiensis strains the peptides of interest were generated. Chapter 4 describes a sterile enzymatic method to generate peptides from casein. Bioinformatic tools were used to predict enzymes capable of liberating caseicin peptides from casein. Hydrolysates were generated using suitable enzymes, examined and some were found to produce peptides with activity against Cronobacter spp.. This study establishes a potential industrial-grade method to generate antimicrobial peptides. Administration of GLP-1 leads to improved glycaemic control in diabetes patients. Generation of a recombinant lactic acid bacteria capable of producing a GLP-1 analogue is described in Chapter 5. In-vivo analysis confirmed insulinotropic activity. The results illustrate a method using bacteriocin producing cellular machinery to generate bioactive peptides. This thesis describes the generation of bioactive peptides by bacterial fermentation, tailored enzymatic hydrolysis and recombinant bacterial methods. The techniques described contribute to bioactive peptide research with regards novel methods of production and industrial scale-up.

Monitoring and modelling the morphodynamic evolution of a breached barrier beach system

Predicting the evolution of a coastal cell requires the identification of the key drivers of morphology. Soft coastlines are naturally dynamic but severe storm events and even human intervention can accelerate any changes that are occurring. However, when erosive events such as barrier breaching occur with no obvious contributory factors, a deeper understanding of the underlying coastal processes is required. Ideally conclusions on morphological drivers should be drawn from field data collection and remote sensing over a long period of time. Unfortunately, when the Rossbeigh barrier beach in Dingle Bay, County Kerry, began to erode rapidly in the early 2000’s, eventually leading to it breaching in 2008, no such baseline data existed. This thesis presents a study of the morphodynamic evolution of the Inner Dingle Bay coastal system. The study combines existing coastal zone analysis approaches with experimental field data collection techniques and a novel approach to long term morphodynamic modelling to predict the evolution of the barrier beach inlet system. A conceptual model describing the long term evolution of Inner Dingle Bay in 5 stages post breaching was developed. The dominant coastal processes driving the evolution of the coastal system were identified and quantified. A new methodology of long term process based numerical modelling approach to coastal evolution was developed. This method was used to predict over 20 years of coastal evolution in Inner Dingle Bay. On a broader context this thesis utilised several experimental coastal zone data collection and analysis methods such as ocean radar and grain size trend analysis. These were applied during the study and their suitability to a dynamic coastal system was assessed.

Irish state infrastructural investment: an analysis of past patterns, and an outline of a future integrated systems of systems evaluation methodology

The literature clearly links the quality and capacity of a country’s infrastructure to its economic growth and competitiveness. This thesis analyses the historic national and spatial distribution of investment by the Irish state in its physical networks (water, wastewater and roads) across the 34 local authorities and examines how Ireland is perceived internationally relative to its economic counterparts. An appraisal of the current status and shortcomings of Ireland’s infrastructure is undertaken using key stakeholders from foreign direct investment companies and national policymakers to identify Ireland's infrastructural gaps, along with current challenges in how the country is delivering infrastructure. The output of these interviews identified many issues with how infrastructure decision-making is currently undertaken. This led to an evaluation of how other countries are informing decision-making, and thus this thesis presents a framework of how and why Ireland should embrace a Systems of Systems (SoS) methodology approach to infrastructure decision-making going forward. In undertaking this study a number of other infrastructure challenges were identified: significant political interference in infrastructure decision-making and delivery the need for a national agency to remove the existing ‘silo’ type of mentality to infrastructure delivery how tax incentives can interfere with the market; and their significance. The two key infrastructure gaps identified during the interview process were: the need for government intervention in the rollout of sufficient communication capacity and at a competitive cost outside of Dublin; and the urgent need to address water quality and capacity with approximately 25% of the population currently being served by water of unacceptable quality. Despite considerable investment in its national infrastructure, Ireland’s infrastructure performance continues to trail behind its economic partners in the Eurozone and OECD. Ireland is projected to have the highest growth rate in the euro zone region in 2015 and 2016, albeit that it required a bailout in 2010, and, at the time of writing, is beginning to invest in its infrastructure networks again. This thesis proposes the development and implementation of a SoS approach for infrastructure decision-making which would be based on: existing spatial and capacity data of each of the constituent infrastructure networks; and scenario computation and analysis of alternative drivers eg. Demographic change, economic variability and demand/capacity constraints. The output from such an analysis would provide valuable evidence upon which policy makers and decision makers alike could rely, which has been lacking in historic investment decisions.

The development of an algorithm to track blood pressure accurately based on the pulse transit time method

Oscillometric blood pressure (BP) monitors are currently used to diagnose hypertension both in home and clinical settings. These monitors take BP measurements once every 15 minutes over a 24 hour period and provide a reliable and accurate system that is minimally invasive. Although intermittent cuff measurements have proven to be a good indicator of BP, a continuous BP monitor is highly desirable for the diagnosis of hypertension and other cardiac diseases. However, no such devices currently exist. A novel algorithm has been developed based on the Pulse Transit Time (PTT) method, which would allow non-invasive and continuous BP measurement. PTT is defined as the time it takes the BP wave to propagate from the heart to a specified point on the body. After an initial BP measurement, PTT algorithms can track BP over short periods of time, known as calibration intervals. After this time has elapsed, a new BP measurement is required to recalibrate the algorithm. Using the PhysioNet database as a basis, the new algorithm was developed and tested using 15 patients, each tested 3 times over a period of 30 minutes. The predicted BP of the algorithm was compared to the arterial BP of each patient. It has been established that this new algorithm is capable of tracking BP over 12 minutes without the need for recalibration, using the BHS standard, a 100% improvement over what has been previously identified. The algorithm was incorporated into a new system based on its requirements and was tested using three volunteers. The results mirrored those previously observed, providing accurate BP measurements when a 12 minute calibration interval was used. This new system provides a significant improvement to the existing method allowing BP to be monitored continuously and non-invasively, on a beat-to-beat basis over 24 hours, adding major clinical and diagnostic value.

Floating wave energy converters: wave measurement & analysis techniques

The wave energy industry is entering a new phase of pre-commercial and commercial deployments of full-scale devices, so better understanding of seaway variability is critical to the successful operation of devices. The response of Wave Energy Converters to incident waves govern their operational performance and for many devices, this is highly dependent on spectral shape due to their resonant properties. Various methods of wave measurement are presented, along with analysis techniques and empirical models. Resource assessments, device performance predictions and monitoring of operational devices will often be based on summary statistics and assume a standard spectral shape such as Pierson-Moskowitz or JONSWAP. Furthermore, these are typically derived from the closest available wave data, frequently separated from the site on scales in the order of 1km. Therefore, variability of seaways from standard spectral shapes and spatial inconsistency between the measurement point and the device site will cause inaccuracies in the performance assessment. This thesis categorises time and frequency domain analysis techniques that can be used to identify changes in a sea state from record to record. Device specific issues such as dimensional scaling of sea states and power output are discussed along with potential differences that arise in estimated and actual output power of a WEC due to spectral shape variation. This is investigated using measured data from various phases of device development.