Phytochemical studies and biological activity of carnivorous plants from the Mediterranean region

Tese de doutoramento, Ciências Biotecnológicas (Biotecnologia Vegetal), Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2010

Marine foundation species shifting ranges and genetic baselines

This thesis revealed the most importance factors shaping the distribution, abundance and genetic diversity of four marine foundation species. Environmental conditions, particularly sea temperatures, nutrient availability and ocean waves, played a primary role in shaping the spatial distribution and abundance of populations, acting on scales varying from tens of meters to hundreds of kilometres. Furthermore, the use of Species Distribution Models (SDMs) with biological records of occurrence and high-resolution oceanographic data, allowed predicting species distributions across time. This approach highlighted the role of climate change, particularly when extreme temperatures prevailed during glacial and interglacial periods. These results, when combined with mtDNA and microsatellite genetic variation of populations allowed inferring for the influence of past range dynamics in the genetic diversity and structure of populations. For instance, the Last Glacial Maximum produced important shifts in species ranges, leaving obvious signatures of higher genetic diversities in regions where populations persisted (i.e., refugia). However, it was found that a species’ genetic pool is shaped by regions of persistence, adjacent to others experiencing expansions and contractions. Contradicting expectations, refugia seem to play a minor role on the re(colonization) process of previously eroded populations. In addition, the available habitat area for expanding populations and the inherent mechanisms of species dispersal in occupying available habitats were also found to be fundamental in shaping the distributions of genetic diversity. However, results suggest that the high levels of genetic diversity in some populations do not rule out that they may have experienced strong genetic erosion in the past, a process here named shifting genetic baselines. Furthermore, this thesis predicted an ongoing retraction at the rear edges and extinctions of unique genetic lineages, which will impoverish the global gene pool, strongly shifting the genetic baselines in the future.

Recreational shore fishing in southern Portugal: biological and socio-economic aspects and perspectives for management

Although overfishing is a concern for many fish stocks, it was for a long time only associated with commercial fishing exploitation, with less or no attention being given to the recreational fisheries. Recent research has shown however that the impact of recreational fishing on particular species can be considerable, and that the recreational harvest needs to be taken into account if fisheries are to be accurately assessed and effectively managed. In Portugal, the first recreational fishing regulations were only recently implemented. However, mirroring other European countries, regulations lacked scientific support, and specific knowledge of the activity was limited to a few studies with limited coverage. This thesis aimed to characterize the biological and socioeconomic aspects of the recreational shore angling activity in southern Portugal, to investigate whether the regulations in place were adequate and effective, and to provide recommendations for improved management and conservation of the inshore fisheries resources. A combined aerial-roving survey was conducted to gather data on fishing effort, catch, fishing trips and socioeconomic aspects (including anglers’ perceptions of regulations) of the recreational angling activity. The analysis of anglers’ catches suggested that compliance with daily bag limits was high, with less than 0.5% of creels exceeding the 10 kg angler-1 day-1 bag limit. Overall, 11.5% of the retained fishes were undersized, but non-compliance with minimum size limits was found to be high for some species (e.g. seabass, 73% undersized). In terms of the impact of recreational shore angling, the total estimated catches corresponded to less than 1% of the commercial landings for the same period (shared species). However, shore angling catches for white sea bream (Diplodus sargus) were found to be considerable, corresponding to 65% of the commercial landings (39.4% of total catch). In terms of anglers’ perceptions about the recreational fishing regulations in Portugal, the present study has shown that the majority of anglers accepted the existence of some kind of SRF regulations, but in general there was a partial or total disagreement with the recreational fishing restrictions recently put in place. Most anglers perceived themselves as not being involved in the decision-making process and claimed that some restrictions lacked a meaningful rationale (e.g. prohibition of fishing from piers/jetties). Fishers’ awareness with regard to specific aspects of the restrictions (such as the rationale for minimum size limits) was found to be very limited. During the same period, catches from sport fishing competitions were examined to test for differences with the recreational activity in terms of catches, and evaluate long term trends in catch and mean size of fish. Catches of the sport fishing competitions were found to be different from those observed for recreational fishing, being dominated by different species (e.g. garfish, mullets), and suggesting different fishing strategies of the the two types of anglers. High percentages of undersized fish were observed to be captured (and retained) during the competitions (in particular seabass, with 100% undersized), probably as a result of a single allowable minimum size (AMS) of 15 cm for all species in use in competitions. Lastly, catch and release fishing experiments were carried out to assess post-release mortality of three recreationally important species: two banded sea bream Diplodus vulgaris; black sea bream Spondyliosoma cantharus; and gilthead sea bream Sparus aurata. Post-release mortalities were found to be low (0-12%). The main predictor of mortality for Sparus aurata was anatomical hooking location, with 63% of the fishes that died being deeply hooked. The results support the release of fish, either from mandatory (e.g. minimum landing sizes) or voluntary practices. In summary, this thesis has demonstrated that the impact of recreational fishing for particular species is significant and needs to be taken into account for more effective management and stock assessment purposes. It has also highlighted several management issues that should be addressed in order to promote more adequate regulations in the future and prevent noncompliance issues. A periodic monitoring of the recreational fishing activity including all fishing modes (i.e. spear fishing, boat, and shore angling) would also be beneficial to ensure a timely knowledge on the global recreational fishing activity and support future management actions.

Biological membranes : biophysical properties and aquaporin function

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

A Community-Level, Mesoscale Analysis of Fish Assemblage Structure in Shoreline Habitats of a Large River Using Multivariate Regression Trees.

The mesoscale (100–102 m) of river habitats has been identified as the scale that simultaneously offers insights into ecological structure and falls within the practical bounds of river management. Mesoscale habitat (mesohabitat) classifications for relatively large rivers, however, are underdeveloped compared with those produced for smaller streams. Approaches to habitat modelling have traditionally focused on individual species or proceeded on a species-by-species basis. This is particularly problematic in larger rivers where the effects of biological interactions are more complex and intense. Community-level approaches can rapidly model many species simultaneously, thereby integrating the effects of biological interactions while providing information on the relative importance of environmental variables in structuring the community. One such community-level approach, multivariate regression trees, was applied in order to determine the relative influences of abiotic factors on fish assemblages within shoreline mesohabitats of San Pedro River, Chile, and to define reference communities prior to the planned construction of a hydroelectric power plant. Flow depth, bank materials and the availability of riparian and instream cover, including woody debris, were the main variables driving differences between the assemblages. Species strongly indicative of distinctive mesohabitat types included the endemic Galaxias platei. Among other outcomes, the results provide information on the impact of non-native salmonids on river-dwelling Galaxias platei, suggesting a degree of habitat segregation between these taxa based on flow depth. The results support the use of the mesohabitat concept in large, relatively pristine river systems, and they represent a basis for assessing the impact of any future hydroelectric power plant construction and operation. By combing community classifications with simple sets of environmental rules, the multivariate regression trees produced can be used to predict the community structure of any mesohabitat along the reach.

Analysis of zooplankton species composition and depth-based habitat preference in Nootka Sound, B.C., Canada

Senior thesis written for Oceanography 445

Mapping multi-species habitat use for marine conservation planning

Tese de Doutoramento, Ciências do Mar (Biologia Marinha)

Cobalt complexes with pyrazole ligands as catalyst precursors for the peroxidative oxidation of cyclohexane: Z-ray absorption spectroscopy studies and biological applications

[CoCl(-Cl)(Hpz(Ph))(3)](2) (1) and [CoCl2(Hpz(Ph))(4)] (2) were obtained by reaction of CoCl2 with HC(pz(Ph))(3) and Hpz(Ph), respectively (Hpz(Ph)=3-phenylpyrazole). The compounds were isolated as air-stable solids and fully characterized by IR and far-IR spectroscopy, MS(ESI+/-), elemental analysis, cyclic voltammetry (CV), controlled potential electrolysis, and single-crystal X-ray diffraction. Electrochemical studies showed that 1 and 2 undergo single-electron irreversible (CoCoIII)-Co-II oxidations and (CoCoI)-Co-II reductions at potentials measured by CV, which also allowed, in the case of dinuclear complex 1, the detection of electronic communication between the Co centers through the chloride bridging ligands. The electrochemical behavior of models of 1 and 2 were also investigated by density functional theory (DFT) methods, which indicated that the vertical oxidation of 1 and 2 (that before structural relaxation) affects mostly the chloride and pyrazolyl ligands, whereas adiabatic oxidation (that after the geometry relaxation) and reduction are mostly metal centered. Compounds 1 and 2 and, for comparative purposes, other related scorpionate and pyrazole cobalt complexes, exhibit catalytic activity for the peroxidative oxidation of cyclohexane to cyclohexanol and cyclohexanone under mild conditions (room temperature, aqueous H2O2). Insitu X-ray absorption spectroscopy studies indicated that the species derived from complexes 1 and 2 during the oxidation of cyclohexane (i.e., Ox-1 and Ox-2, respectively) are analogous and contain a Co-III site. Complex 2 showed low invitro cytotoxicity toward the HCT116 colorectal carcinoma and MCF7 breast adenocarcinoma cell lines.

Ditopic molecular architectures for the recognition of anionic species

Dissertation presented to obtain the Ph.D degree in Chemistry

Biological colonization on majolica glazed tiles: biodeterioration, bioreceptivity and mitigation strategies

The impact of microbial activity on the deterioration of cultural heritage is a well-recognized global problem. Glazed wall tiles constitute an important part of the worldwide cultural heritage. When exposed outdoors, biological colonization and consequently biodeterioration may occur. Few studies have dealt with this issue, as shown in the literature review on biodiversity, biodeterioration and bioreceptivity of architectural ceramic materials. Due to the lack of knowledge on the biodeteriogens affecting these assets, the characterization of microbial communities growing on Portuguese majolica glazed tiles, from Pena National Palace (Sintra, Portugal) and another from Casa da Pesca (Oeiras, Portugal) was carried out by culture and molecular biology techniques. Microbial communities were composed of microalgae, cyanobacteria, bacteria and fungi, including a new fungal species (Devriesia imbrexigena) described for the first time. Laboratory-based colonization experiments were performed to assess the biodeterioration patterns and bioreceptivity of glazed wall tiles produced in laboratory. Microorganisms previously identified on glazed tiles were inoculated on pristine and artificially aged tile models and incubated under laboratory conditions for 12 months. Phototrophic microorganisms were able to grow into glaze fissures and the tested fungus was able to form oxalates over the glaze. The bioreceptivity of artificially aged tiles was higher for phototrophic microorganisms than pristine tile models. A preliminary approach on mitigation strategies based on in situ application of commercial biocides and titanium dioxide (TiO2) nanoparticles on glazed tiles demonstrated that commercial biocides did not provide long term protection. In contrast, TiO2 treatment caused biofilm detachment. In addition, the use of TiO2 thin films on glazed wall tiles as a protective coating to prevent biological colonization was analysed under laboratorial conditions. Finally, conservation notes on tiles exposed to biological colonization were presented.

Promoter rearrangements cause species-specific hepatic regulation of the glyoxylate reductase/hydroxypyruvate reductase gene by the peroxisome proliferator-activated receptor alpha.

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor alpha (PPARalpha) of the mouse GRHPR gene in liver. Mice fed with a PPARalpha ligand or in which PPARalpha activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARalpha present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARalpha ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARalpha-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARalpha regulation. Overall, these data indicate a species-specific regulation by PPARalpha of GRHPR, a key gene of the glyoxylate cycle.

Gastric supply manipulation to modulate ghrelin production and enhance vascularization to the cardia: proof of the concept in a porcine model.

Introduction. Selective embolization of the left-gastric artery (LGA) reduces levels of ghrelin and achieves significant short-term weight loss. However, embolization of the LGA would prevent the performance of bariatric procedures because the high-risk leakage area (gastroesophageal junction [GEJ]) would be devascularized. Aim. To assess an alternative vascular approach to the modulation of ghrelin levels and generate a blood flow manipulation, consequently increasing the vascular supply to the GEJ. Materials and methods. A total of 6 pigs underwent a laparoscopic clipping of the left gastroepiploic artery. Preoperative and postoperative CT angiographies were performed. Ghrelin levels were assessed perioperatively and then once per week for 3 weeks. Reactive oxygen species (ROS; expressed as ROS/mg of dry weight [DW]), mitochondria respiratory rate, and capillary lactates were assessed before and 1 hour after clipping (T0 and T1) and after 3 weeks of survival (T2), on seromuscular biopsies. A celiac trunk angiography was performed at 3 weeks. Results. Mean (±standard deviation) ghrelin levels were significantly reduced 1 hour after clipping (1902 ± 307.8 pg/mL vs 1084 ± 680.0; P = .04) and at 3 weeks (954.5 ± 473.2 pg/mL; P = .01). Mean ROS levels were statistically significantly decreased at the cardia at T2 when compared with T0 (0.018 ± 0.006 mg/DW vs 0.02957 ± 0.0096 mg/DW; P = .01) and T1 (0.0376 ± 0.008mg/DW; P = .007). Capillary lactates were significantly decreased after 3 weeks, and the mitochondria respiratory rate remained constant over time at the cardia and pylorus, showing significant regional differences. Conclusions. Manipulation of the gastric flow targeting the gastroepiploic arcade induces ghrelin reduction. An endovascular approach is currently under evaluation.

Characterization and application of two RANK-specific antibodies with different biological activities.

Antibodies play an important role in therapy and investigative biomedical research. The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation and epithelial cell activation. However, the study of RANK biology has been hampered by missing or insufficient characterization of high affinity tools that recognize RANK. Here, we present a careful description and comparison of two antibodies, RANK-02 obtained by phage display (Newa, 2014 [1]) and R12-31 generated by immunization (Kamijo, 2006 [2]). We found that both antibodies recognized mouse RANK with high affinity, while RANK-02 and R12-31 recognized human RANK with high and lower affinities, respectively. Using a cell apoptosis assay based on stimulation of a RANK:Fas fusion protein, and a cellular NF-κB signaling assay, we showed that R12-31 was agonist for both species. R12-31 interfered little or not at all with the binding of RANKL to RANK, in contrast to RANK-02 that efficiently prevented this interaction. Depending on the assay and species, RANK-02 was either a weak agonist or a partial antagonist of RANK. Both antibodies recognized human Langerhans cells, previously shown to express RANK, while dermal dendritic cells were poorly labeled. In vivo R12-31 agonist activity was demonstrated by its ability to induce the formation of intestinal villous microfold cells in mice. This characterization of two monoclonal antibodies should now allow better evaluation of their application as therapeutic reagents and investigative tools.

Trends in fish prey capture by common terns following zebra mussel invasion : do terns track fish species abundance? /

The Common Tern (Sterna hirundo) is a ground nesting colonial seabird. Terns rely primarily on small prey fishes which they obtain through plunge diving for their survival as well as the survival of their offspring during the breeding season. The zebra mussel (Dreissena polymorpha) is a small bivalve mollusk that invaded North American waters in the late 1980's. Through its suspension feeding, the zebra mussel has the ability to alter the entire aquatic ecosystem, ultimately leading to a reduction in pelagic organisms including small prey fish. The objective of the study was to determine what (if any) indirect effects the invasion of the zebra mussel has had on fish prey captured by terns. The study took place in two separate two-year periods, 1990-91 and 1995-96 on a concrete breakwall off the north shore of Lake Erie near Port Colborne, Ontario. Daily nest checks revealed clutch initiation dates, egg-laying chronology, hatching success and morphological egg characteristics (length and breadth). Behavioural observations included time each sex spent in attendance with its brood, the frequency of feeding chicks and the prey species composition and size fed to chicks as well as to females (courtship feeding). Egg sizes did not differ between study periods, nor did feeding rates to chicks, suggesting that food was not a limiting resource. Terns spent less time with their broods (more time foraging) in the 1995-96 period. However, they also had significantly larger broods and fledged more offspring. The time of each individual foraging trip decreased, suggesting that fish were easier to obtain in 1995 and 1996. Lastly, kleptoparasitism rates decreased, suggesting that the costs of foraging (time, energy) actually decreased as fewer birds adopted this strategy to compensate for what I assumed to be a lack of available food (fish). The only significant difference between the periods of 1990, 1991 and 1995, 1996 was a change in diet. Terns delivered significantly fewer rainbow smelt and more emerald shiner in 1995 and 1996. However, the average size of fish delivered did not change. Thus, there was little impact on prey captured by Common Terns in Lake Erie since the invasion of the zebra mussel.

Genetic variation and evolutionary divergence within and among populations, species, and genera of the Cambarinae

Seven crayfish species from three genera of the subfamily Cambarinae were electrophoretically examined for genetic variation at a total of twenty-six loci. Polymorphism was detected primarily at three loci: Ao-2, Lap, and Pgi. The average heterozygosities over-all loci for each species were found to be very low when compared to most other invertebrate species that have been examined electrophoretically. With the exception of Cambarus bartoni, the interpopulation genetic identities are high within any given species. The average interspecific identities are somewhat lower and the average intergeneric identities are lower still. Populations, species and genera conform to the expected taxonomic progression. The two samples of ~ bartoni show high genetic similarity at only 50 percent of the loci compared. Locus by locus identity comparisons among species yield U-shaped distributions of genetic identities. Construction of a phylogenetic dendrogram using species mean genetic distances values shows that species grouping is in agreement with morphological taxonomy with the exception of the high similarity between Orconectespropinquus and Procambarus pictus. This high similarity suggests the possibility of a regulatory change between the two species. It appears that the low heterozygosities, high interpopulation genetic identities, and taxonomic mispositioning can all be explained on the basis of low mutation rates.