Dissertations / Theses on the topic 'Biological tags'

A design of a novel biological assay platform is presented in this thesis for the application of high throughput DNA sequencing and multiplexing biological assays. Digital magnetic carriers are responsible for the labelling and carrying of biological molecules or DNA strands through the microfluidic cells for various chemical reaction and analysis. This carrier design contains a bit addressable “magnetic barcode” made of patterned magnetic thin film (e.g. Permalloy or Cobalt), sandwiched in between two rigid SU8 polymer protective layers. I utilized magnetic elements with strong shape anisotropy and different coercive fields as the data storage elements, so each one of the magnetic barcodes elements can be addressed individually by a single globally applied variable magnetic field. The main idea being that all the carriers can be made using only a single photolithography template, which is particularly suitable for low cost mass production. The “label” or “signatures” can be coded or re-coded to the carriers in real time as and when needed. This approach is ideal for labelling the oligonucleotides during the split-and-mix synthesis or for the combinatorial library-based high-throughput multiplexed bioassays. I describe how the design of the magnetic barcode is optimized by engineering the coercivity of each barcodes element, allowing the number of available signatures to be increased. I also show that by using a globally applied magnetic field and the magneto-optical Kerr microscope, the magnetic elements in the multi-bit magnetic carriers can be addressed individually and encoded/decoded remotely. The power of this approach is the read/write technique, which allows modest globally applied magnetic fields to write almost unlimited numbers of codes to a large population of carriers rather than individuals.

Four hundred papers published in refereed papers during the years 1935-1985 were researched for information on the biology of trypanorhynchs, and a publishable host-parasite list was compiled for this period as a complement to the only previous monograph (published forty-five years ago) on all known members of the order Trypanorhyncha (Platyhelminthes: Cestoda). The theoretical information gained from this initial exercise was then used in carrying out original research on trypanorhynchs found in over 1,000 elasmobranch and teleost fish. This led to a choice of three research topics for more detailed investigations: (i) taxonomic studies on four little-known species and the application of this information to fisheries biology, (ii) life-cycle studies on Grillotia erinaceus (van Beneden, 1858) and (iii) the use of Grillotia smaris-gore (Wagener, 1854) Dollfus, 1946 as a biological tag for commercially important teleosts. A pre-requisite of this last problem was the need to review critically all information on the use of parasites as population indicators. Thus a further 100 papers published in primary journals were researched. The work concludes with a discussion of the above and also brief comments on the need for further research on tapeworms of the order Trypanorhyncha as an aid to our understanding of other basic contemporary problems in parasitology, relating to host-specificity, ecology and phylogeny.

Recently, questions have been raised as to the appropriateness of stock boundaries in the Atlantic horse mackerel (Trachurus trachurus) and the herring (Clupea harengus) in European waters. To this end, 1919 horse mackerel were examined for parasitic infections from samples taken at nineteen sites around Europe. Forty-five species of parasites were recorded, eleven of which are new host records, and one may be a new species. A number of these are suitable for use as biological tags. Results showed a significant degree of separation between fish from the North Sea and Western stocks, based on the relative abundance of the nematodes Aniaskis spp. and Hysterothylacium sp. The endoparasitic fauna of 4033 herring at various life stages from sites to the west of the British Isles, the North Sea, Baltic Sea and northern Norway were examined to obtain information on stock identity, mixing and recruitment patterns in these areas. Results revealed that substantial stock mixing takes place to the west of the British Isles, with fish from a wide area being found together in non-spawning aggregations off the Hebrides. This area is also home at different times of year to two different spawning populations of fish which recruit from nursery grounds in the eastern North Sea and from the west of Scotland. Investigations were made of the usefulness of the ribosomal small sub-unit cytochrome oxidase (I) gene of the parasitic nematode worm, Anisakis simplex s.s., as a tag of host population biology. Results revealed the COI gene to be highly variable, with around 50% of worms sequenced having unique haplotypes. There were no population structures evident from analysis of genetic distances.

Parasites are a ubiquitous but often unseen, unacknowledged and understudied component of biological communities. However, their roles in structuring ecosystems, their influence on the evolutionary history of host species and their potential applications are slowly being uncovered. The snoek, Thyrsites atun, is a nomadic predator native to the cold coastal waters of the southern Hemisphere. Being a major target of the South African inshore line-fishery, the snoek is a socioeconomically important species whose ecological significance in the southern Benguela should not be underestimated. This study aimed to survey the metazoan parasite community of snoek off South Africa, assess host-parasite relationships and evaluate the potential of parasites as biological tags for stock structure studies. Examination of 210 snoek (FL 411 - 1040 mm) revealed them to be host to 16 parasite taxa. These included 9 new host records (Tentacularia coryphaenae, Caligus coryphaenae, Caligus dakari, Corynosoma australe, Nothobomolochus fradei, Hatschekia conifera, Bolbosoma vasculosum, Rhadinorhynchus cadenati, Digenea sp.) and 4 new locality records (Molicola uncinatus, Pseudoterranova sp., C. dakari, B. vasculosum). A further three cosmopolitan taxa (Anisakis sp., Kudoa thyrsites, Hepatoxylon trichiuri) as well as Caligus zei were also recorded.

Glucosyltransferases catalyze glucosylation by transferring glucose from UDP-activated sugar donor to the acceptor substrates. This research is focused on the study of the effect of a single point mutation on enzyme activity, characterization of a flavonol specific 3-Oglucosyltransferase (Cp-3-O-GT) mutant- P145T, and further modification of the clone to cleave off tags from recombinant wild type and P145T mutant proteins in order to crystallize the proteins. Multiple sequence alignment and homology modeling was done to identify candidate residues for mutation. Cp-3-O-GT was modeled with a flavonoid 3-O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT- P145T mutant to test the hypothesis that that mutation of proline by threonine in Cp-3-O-GT could alter substrate or regiospecificity of Cp-3-O-GT. While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to flavonols. This is significant because flavanones and flavones do not contain a 3-OH group. HPLC was performed to identify the reaction products. Early results indicated that the mutant protein glucosylates naringenin at the 7-OH position forming prunin. Results are being used to revisit and refine the structure model. In other related work, a thrombin cleavage site was inserted into wild type and recombinant P145Tenzyme and we are currently working on transformation into yeast for recombinant protein expression. Cleaving off tags is a pre-requisite to future efforts to crystallize the proteins. Solving the crustal structures will make a significant contribution to the structural and functional study of plant flavonoid GTs in general and Cp-3-O-GT in particular.

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Large hydroelectric dams along the Paraná River have caused severe impacts on fish communities as they represent obstacles to long-range migratory species that need to migrate to complete their life cycle. Dams can bring numerous problems through the interception of migratory routes and reduction of spawning areas. The most common measures used to mitigate the effect of these physical obstacles is the implementation of fish ladders. It is not only important to implement these structures, but also to monitor them. This study was performed at the Engenheiro Sergio Motta Hydroelectric Power plant in the Upper Paraná River during December/2012 and March/2016. The aim of it was to evaluate the effectiveness of this structure in relation to the attractiveness rate and passage efficiency of to two Neotropical fish species in the fish ladder. In total, 563 fish of genus Leporinus (447 Leporinus obtusidens and 116 Leoporinus piavussu) were tagged and released with the implementation of 32-mm programmable transponders (PIT-tags). Radio Frequency Identification (RFID) was used to monitor the system. The attractiveness of the ladder was 8.7%. The minimum time to find it was 1.49 days for the fish released upstream and the maximum time was 449.86 days for the individuals released downstream. The release site had a significant influence on the entry rate of fish released on the other side of the river. No detection was observed for fish released downstream and upstream on the right bank of the river. Only the fish released on the same site where the fish ladder is located were detected, suggesting low attractiveness of the ladder in relation to the studied species. In the course of this study it was verified that for the two species studied it would be extremely important to increase the attractiveness at the entrance of the ladder, as well as to build a second ladder for fish on the right side of the river.
As grandes hidrelétricas ao longo do Rio Paraná têm causado impactos severos nas comunidades de peixes, pois representam obstáculos para as espécies migradoras de longa distância, que precisam migrar para completar seu ciclo de vida. As barragens podem trazer inúmeros problemas por meio da intercepção das rotas migratórias e redução das áreas de desova. As medidas mais comuns utilizadas para mitigar o efeito desses obstáculos físicos é a implementação de escadas para peixes. Não é somente importante a implementação dessas estruturas, como também o monitoramento. Esse estudo realizado na escada para peixes da Usina Hidrelétrica Engenheiro Sérgio Motta no alto Rio Paraná no período de dezembro/2012 a março/2016 teve como objetivo avaliar a efetividade dessa estrutura em relação a taxa de atratividade e eficiência de passagem para duas espécies de peixes migradores neotropicais. No total, foram marcados e liberados 563 peixes do gênero Leporinus (447 Leporinus obtusidens e 116 Leoporinus piavussu) com a implantação de transponders com códigos programáveis (PIT-tags) de 32 mm. Foi utilizado o sistema de rádio frequência (RFID Radio Frequency Identification) para o monitoramento do sistema. A atratividade da escada foi de 8,7%. O tempo mínimo para encontrar a escada correspondeu a 1,49 dias para os peixes liberados a montante e o tempo máximo foi de 449,86 dias para os indivíduos liberados a jusante. O local de liberação influenciou significativamente a taxa de entrada dos peixes liberados na margem oposta do rio, sendo que nenhuma detecção foi observada para peixes liberados na margem direita, tanto a jusante como a montante. Foram detectados apenas os peixes liberados na mesma margem onde a escada para peixes está localizada, sugerindo baixa atratividade da escada para as espécies analisadas. No decorrer deste estudo verificou-se que para as duas espécies estudadas seria extremamente importante aumentar a atratividade na entrada da escada, como também a construção de uma segunda escada para peixes no lado direito do rio.

Malaria is a disease that is caused by parasite called Plasmodium spp. and trasmitted by female Anopheles mosquitoes to the host. The disease has great impact around the world and there are half a million deaths and several hundred million infections every year. Studies revealed that there are two actin isoforms in the parasite, actin I and actin II. Absence of actin II has severe effect on the development of the parasite in the mosquito but the molecular function is still unknown. Identification of interacting proteins is of great importance to understand further the function of the protein. To achieve this goal actin II has to be enriched and this required a tagged version of the protein. In this project purification of the protein was to be achieved through biotinylation. In this method the protein of interest is biotinylated by BirA ligase in the cell and is then purified by , streptavidin. The project involved transfection of vector for Plasmodium berghei, containing the BirA gene and a stage-specific promoter (cdpk4). The construct was integrated in the chromosomal locus Sil6 and introduced to wild-type and actin II knock out parasites. Genotyping by PCR revealed integration of the insert in wild type parasites and phenotypic anaylsis showed no difference between BirA wild type and wild type control parasites. The expression of the BirA ligase in the parasite was investigated with Western blot but no signal was detected.

Triacylglycerol (TAG) is a main storage lipid in seeds and is utilized for seed germination. Humans commonly consume TAG in the form of vegetable oil. Currently, TAG contributes to 25% of the total calories consumed in the developed countries and dependency for biofuel production from plants lipid is also increasing day by day. By 2030 the expected consumption level of vegetable oil will be double and to fulfill this demand it is necessary to increase the oil production in plants. To accomplish this goal, it is pertinent to understand the regulation of TAG accumulation in plants. Avocado (Persea americana), a basal angiosperm, stores up to 70% oil in the form of TAG in nonseed tissue (mesocarp). Here, we use it avocado as a model system to understand TAG biosynthesis in tissues other than seed. In plants, TAG biosynthesis may involve acyl-CoA–dependent or –independent pathway catalyzed by diacylglycerol acyltransferases (DGAT) and phospholipid: diacylglycerol acyltransferases (PDAT), respectively. RNA-Seq and Q-PCR analysis of developing mesocarp of avocado revealed higher expression for DGAT1 and PDAT1, coinciding with the period of TAG accumulation. Therefore, we hypothesize that DGAT1 and PDAT1 are responsible for catalyzing the terminal step in TAG biosynthesis in Avocado mesocarp. Using the transcriptome data, we identified full-length coding sequences for DGAT1 and PDAT1. These acyltransferases are being cloned and will be characterized for their enzyme activity and substrate specificity, subsequent to their expression in yeast. Complementation of Arabidopsis dgat1 and pdat1 mutants and phenotypic characterization will also be carried out using Gateway-cloning techniques and transgenic lines will be assayed for TAG content in seeds. This study is expected to provide basic understanding of TAG accumulation in avocado mesocarp tissue.

Les comunitats de paràsits de peixos han estat usades com a eines integrals per a la salut de l’ecosistema. L’objectiu general de la present tesi és doble: d’una banda es pretenia proporcionar una millor comprensió de la composició i l’estructura de les comunitats parasítiques dels teleostis perciformes Mullus barbatus L., Spicara maena (L.) i Trachinus draco L. del nord-oest Mediterrani (especialment, la costa de Catalunya) i testar si les variacions en l’estructura de la comunitat parasítica podien estar relacionades amb les càrregues de pol·lució i/o amb la varietat natural (geogràfica i temporal). El mostreig va tenir lloc el 2007 al nord-oest del Mar Mediterrani, davant de la costa de Barcelona (Catalunya, nord-est d’Espanya) a la plataforma continental, a profunditats d’entre 50-68 metres. Una vegada al vaixell, els individus foren mesurats (longitud total) i pesats (pes total). Els espècimens foren immediatament congelats a -20ºC en bosses de plàstic individuals per a posteriors procediments al laboratori. Els individus descongelats foren processats i examinats per buscar ectoparàsits i endoparàsits sota l’estereomicroscopi. Tots els paràsits recollits foren comptats i processats seguint les tècniques parasitològiques. Una nova espècie críptica pertanyent al “complexe A. laguncula”, Aponurus mulli n. sp., fou descrita en base a l’abundant material trobat en Mullus barbatus (hoste tipus) i en M. surmuletus capturats prop de les costes Mediterrànies espanyoles mentre que es varen redescriure dos digenis Hemiuridae que són freqüentment citats però molt poc coneguts, en base al material capturat a la costa de Barcelona de l’oest Mediterrani, Lecithochirium musculus (Looss, 1907) (Lecithochiriinae), , i Ectenurus lepidus Looss, 1907 (Dinurinae). D’altra banda, s’examinaren les comunitats parasítiques de Mullus barbatus L., una espècie mediterrània bioindicadora, mostrejada en un gradient de variacions de petita escala de PCBs en els sediments de la plataforma continental. Les respostes dels paràsits als nivells moderats de pol·lució observades foren validades simultàniament amb el monitoratge químic i amb l’efecte dels biomarcadors bioquímics. També vàrem descriure les comunitats de paràsits de Spicara maena (L.) i Trachinus draco L. del nord-oest Mediterrani, amb una visió en l’ús dels paràsits com a marcadors ambientals. El nematode Hysterothylacium fabri fou proposats per a futurs estudis de monitoratge ecològic de la costa de Catalunya.
Parasite communities of fish have been used as comprehensive tags of ecosystem health. The general target of the present thesis is twofold: on the one hand we aimed to provide a better understanding of the composition and structure of parasite communities in the perciform teleosts Mullus barbatus L., Spicara maena (L.) and Trachinus draco L. from the north-western Mediterranean (specifically, the Catalonian coasts); on the other hand we aimed to test whether variations in parasite community structure can be related to pollution loads and/or with natural variability (geographical and temporal). Sampling took place in 2007 in the north-western Mediterranean Sea, in front of the coast of Barcelona (north-eastern Spain) on the continental shelf at depth of 50-68m. Once on board, individuals were measured (total length) and weighted (total weight). They were immediately frozen at -20ºC in an individual plastic bag for posterior procedures in the laboratory. Thawed specimens were processed and examined for ectoparasites and endoparasites under the stereomicroscope. All parasites collected were counted and processed following parasitological procedures. A new cryptic species of the ‘A. laguncula complex’, Aponurus mulli n. sp., was described on the basis of abundant material from Mullus barbatus (type-host) and M. surmuletus off the Spanish Mediterranean coasts whereas two frequently reported but poorly known Hemiuridae digeneans, Lecithochirium musculus (Looss, 1907) (Lecithochiriinae) and Ectenurus lepidus Looss, 1907 (Dinurinae), were redescribed based on material from off the Barcelona coast of the western Mediterranean. Otherwise, parasite communities of the Mediterranean sentinel fish species, Mullus barbatus, sampled at a small-scale PCB gradient at the shelf sediments, were examined. The observed parasite responses to moderate levels of pollution were simultaneously validated by both chemical monitoring and biochemical biomarkers effects. We also described the parasite communities of Spicara maena (L.) and Trachinus draco L. off the north-western Mediterranean, with a view of using parasite species as environmental tags. The nematode Hysterothylacium fabri is purposed for future studies in ecological biomonitoring for the coast of Catalonia.

Transcranial alternating current stimulation (tACS) is a non-invasive brain stimulation method that involves the application of weak electric currents to the scalp. tACS has the potential to be an inexpensive, easily administrable, and well-tolerated multi-purpose tool for cognitive and clinical neuroscience as it could be applied to establish the functional role of rhythmic brain activity, and to treat neural disorders, in particular those where these rhythms have gone awry. However, the mechanisms by which tACS produces both "online" and "offline" effects (that is, those that manifest during stimulation and those that last beyond stimulation offset) are to date still poorly understood. If the potential of tACS is to be harnessed effectively to alter brain activity in a controlled manner, it is fundamental to have a good understanding of how tACS interacts with neuronal dynamics, and of the conditions that promote its effect. This thesis describes three experiments that were conducted to elucidate the mechanisms by which tACS interacts with underlying neural network activity. Experiments 1 and 2 investigated the mechanism by which tACS at alpha frequencies (8 12 Hz, α-tACS) over occipital cortex induces the lasting aftereffects on posterior α power that were previously described in the literature. Two mechanisms have been suggested to underlie alpha power enhancement after α tACS: entrainment of endogenous brain oscillations and/or changes in oscillatory neural networks through spike timing-dependent plasticity (STDP). In Experiment 1, we tested to what extent plasticity can account for tACS-aftereffects when controlling for entrainment characteristics. To this end, we used a novel, intermittent α-tACS protocol and investigated the strength of the aftereffect as a function of phase continuity between successive tACS episodes, as well as the match between stimulation frequency and individual alpha frequency (IAF). Alpha aftereffects were successfully replicated with enhanced α power after intermittent stimulation compared to sham. These aftereffects did not exhibit any of the expected characteristics of prolonged entrainment in that they were independent of tACS phase-continuity and did not show stable phase alignment or synchronisation to the stimulation frequency. These results indicate that prolonged entrainment is insufficient to explain the aftereffects and suggest that the latter emerge through some form of network plasticity. To clarify the nature of these plasticity mechanisms, we then aimed to assess whether STDP could explain the α power increase. We developed a conceptual STDP model that predicted bi-directional changes in α power depending on the relative mismatch between the tACS frequency and IAF. After observing in Experiment 1 that tACS at frequencies slightly lower than the IAF produced α enhancement, Experiment 2 used a similar intermittent protocol that manipulated tACS frequency to be either slightly lower or higher than IAF to respectively enhance or suppress α activity. In addition, a control condition with continuous stimulation aimed to replicate previous results from other groups. However, we did not observe a systematic α power change in any of the active conditions. The lack of consistency between the two experiments raises concerns regarding the reproducibility and effect size of tACS aftereffects. The third experiment investigated the mechanism of online effects and tested predictions that were based on the assumption that entrainment is the underlying process mediating behavioural changes during tACS. We capitalised on two well-described phenomena: firstly, the association between α power lateralisation and visuospatial attention, and secondly, the fluctuation of perceptual performance with α phase. Specifically, the experiment tested whether event-related α-tACS applied over right parieto-occipital cortex can induce a visuospatial bias in a peripheral dot detection task that would reflect α power lateralisation, and whether detection performance depends on the phase of the tACS waveform. In control trials either no tACS or 40 Hz-tACS (gamma) was applied to make use of the putative opposing roles of alpha and gamma oscillations in visual processing. As expected from lateralised enhancement of alpha oscillations, visual detection accuracy was weakly impaired for targets presented in the left visual field, contralateral to tACS. However, this effect was neither frequency specific nor waveform phase-dependent. Therefore, it is unlikely that the negative effect of tACS on visuospatial performance reflects entrainment. Overall, the results of these experiments only partially met our hypotheses. Experiment 1 produced the α enhancement that was expected based on the literature while the follow-up experiment failed to reproduce these results under similar conditions. This outcome demonstrates at best that tACS aftereffects on α activity are not robust, may vary widely across individuals, and might be extremely sensitive to small changes in experimental parameters and state variables. The results of the third experiment call into question the assumption of online entrainment as basis for the observed behavioural effect. These findings point to the need for improved methodology, for more systematic and exhaustive exploration of the relative effects of tACS across different parameter settings, tasks, and individuals; and for the replication of promising but thus far often anecdotal results. They also inspire guidelines for more informative experimental designs.

Glicose e oxigênio são moléculas essenciais para a maioria dos organismos vivos. Além de sua importância nos processos de produção de energia - glicose como fonte de carbono e energia e oxigênio como aceptor dos elétrons doados por NADH e FADH2 - estes dois compostos funcionam como efetuadores, modulando vários processos metabólicos e fisiológicos nas células. Visto que a mitocôndria é um dos alvos afetados pelas disponibilidades destas duas moléculas, nós isolamos e seqüenciamos o genoma mitocondrial de Trichoderma reesei, um fungo multicelular empregado neste trabalho como sistema modelo. Foi estudado o efeito da variação de concentração de glicose e oxigênio sobre a expressão de transcritos do genoma mitocondrial, bem como sua implicação no metabolismo de glicose. São apresentadas análises da expressão gênica de aproximadamente 2000 transcritos de T. reesei submetido a concentrações limitantes de oxigênio dissolvido, realizadas com o emprego da técnica de microarrays de cDNA. Pelo menos 330 transcritos foram diferencialmente expressos em função da disponibilidade de oxigênio. Aqueles envolvidos nos processos de síntese protéica e divisão celular foram regulados negativamente, enquanto transcritos relacionados com funções de defesa celular e síntese de RNA foram positivamente regulados. Uma fração substancial de outros genes afetados pela baixa disponibilidade de oxigênio não possui, atualmente, funções celulares conhecidas. Esta observação deve contribuir para a posterior anotação funcional do genoma de T. reesei. Também foram identificados reguladores transcricionais diferencialmente expressos em baixas tensões de oxigênio. O perfil de expressão destes reguladores aponta-os como potenciais candidatos ao envolvimento com a expressão de genes afetados pela disponibilidade de oxigênio.
Glucose and oxygen are essential molecules in most of living organisms. In addition to their importance in production of energy - glucose as a carbon and energy source and oxygen as an acceptor of electrons donated by NADH and FADH2 - both molecules function as effectors modulating various metabolic and physiological processes in the cell. Because one of the targets affected by both molecules is the mitochondrion, we isolated and sequenced the mitochondrial genome of Trichoderma reesei, a multicellular fungus that is used in this study as a model system. The effect of varying the concentration of glucose and oxygen on the expression of the transcripts of the mitochondrial genome, and its implication on the metabolism of glucose, was studied. Gene-wide expression analyses of nearly 2000 transcripts of T. reesei under limited concentration of dissolved oxygen, using cDNA microarry technique, are presented. At least 330 transcripts were differentially expressed with respect to oxygen availability. Those involved in protein synthesis and cell division processes were downregulated, while transcripts involved in cell defense and RNA synthesis were upregulated. A substantive fraction of other anaerobically affected genes have currently unknown cellular roles, and these results should therefore contribute to further functional annotation of the genome. ln addition, we have identified transcriptional regulators that are differentially expressed at a low oxygen tensions. The expression profile of these regulators points them out as potential candidates involved in the expression of genes affected by oxygen availability.

Cyprinus carpio (L. 1758) è un pesce di acqua dolce di importanza rilevante per il carpfishing. Gli studi su accrescimento, età e impatti sull’ecosistema usano metodologie nocive per le carpe (es. otoliti, elettrofishing). In questo studio si è valutata la fattibilità di campionamento tramite carpfishing (innocuo), calcolato l’accrescimento di carpe del lago “Le Ghiarine” tramite lettura di scaglie, taggato le catture e misurata lunghezza e peso. Nelle sessioni di pesca sono state campionate 56 carpe ed effettuata 1 ricattura. Per ogni carpa è stata prelevata 1 scaglia (fenotipo a specchi) o 4 (per regine) e sono state osservate al microscopio per la conta degli annuli. Si è stimata l’età di 34 campioni, di cui 14 presentavano una zona ricostruita e 20 integre. I restanti 22 erano illeggibili a causa della ricostruzione o della cattiva conservazione. Sui dati ottenuti è stata calcolata la VBGF ma il modello non convergeva ed è stato quindi integrato e corretto con dati bibliografici coerenti con la popolazione in esame, ottenendo: 28.6% di età congruenti alla lettura di laboratorio, 21.4% di età modificate per tener conto della rigenerazione non costante ed il 50% completamente ricalcolate. Il range di anni stimati per la rigenerazione delle scaglie va da 1 a 9, con media 5 e deviazione standard 2. L’ottimizzazione della VBGF ha fornito i seguenti parametri: L∞=104.5, K=0.113 e t0= -0.71. I parametri della relazione lunghezza-peso W=aLb, sono b=2.63 (allometria negativa, p-value=0.0002) e a=0.62. Il quadro generale dei costi di fattibilità è di 401.50€ per 4 sessioni di pesca, circa 40€/giorno. Uno studio tramite carpfishing è quindi fattibile solo a lungo termine per ottenere un numero di campioni adeguato. Per la buona conservazione delle scaglie è opportuno l’uso di alcol o formalina per evitarne il deterioramento. Occorre inoltre un dataset che comprenda le taglie piccole per una migliore ottimizzazione dei dati e dei risultati.

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq)
Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)
Os exossomos s?o respons?veis pela comunica??o c?lula-c?lula e podem influenciar na progress?o tumoral, met?stase e efic?cia terap?utica. Dentre as c?lulas capazes de secretar exossomos est?o as c?lulas tumorais e as c?lulas imunes. Sabe-se que a presen?a das c?lulas imunes ? importante para erradicar os tumores. No entanto, achados recentes demonstram que a inflama??o pode promover o crescimento tumoral. Os macr?fagos associados a tumores (TAMs) s?o conhecidos por apresentarem diferentes subtipos, M1 e M2, capazes de secretarem exossomos. O presente estudo se prop?s a observar o comportamento dos exossomos derivados dos TAMs, dos subtipos 1 e 2, frente a cultura de c?lulas humanas SCC-25, HSC-3 e SAS derivadas de CE de l?ngua, por meio da an?lise da capacidade de invas?o, prolifera??o e viabilidade das c?lulas tumorais na presen?a dos exossomos. Observou-se que as microves?culas derivadas dos TAMs apresentam positividade para CD63, caracterizando-as como exossomos. Os exossomos dos TAMs do subtipo M2 foram os ?nicos a apresentarem marca??o para TGF-?, quando em compara??o com os exossomos M1, THP1 e das linhagens celulares de CE, sugerindo que os exossomos M2 podem ser respons?veis pela express?o de TGF-? nas c?lulas tumorais, uma vez que s?o internalizados. Nos ensaios de migra??o, observou-se que as c?lulas SCC-25 em presen?a de meio de cultura DMEM F/12, apresentaram maior capacidade de invas?o frente aos exossomos M2 (p?0,001), para concentra??o de 0,1 ?g/ml. Para as c?lulas HSC-3 e SAS, n?o foi observada rela??o estatisticamente significante entre a presen?a de exossomos cultivados juntamente com as c?lulas tumorais e a capacidade de invas?o celular (p>0,05). Quando os exossomos foram colocados no compartimento inferior do transwell, as c?lulas HSC-3 em presen?a dos exossomos M2 (1,0 ?g/ml) apresentaram maior capacidade de invas?o (p?0,001). O teste de viabilidade demonstrou que as c?lulas HSC-3 tornam-se mais vi?veis frente ? presen?a dos exossomos M2 (p?0,001) na concentra??o de 50 ?g/ml. Para as c?lulas SCC-25, o resultado foi o mesmo (p?0,05). A imunofluoresc?ncia demonstrou a internaliza??o dos exossomos nas linhagens celulares estudadas. Os achados sugerem que a presen?a de exossomos M2, frente ?s culturas de c?lulas de CE de l?ngua, pode ser um campo de pesquisa importante para futuros estudos com terapias-alvo.
Squamous cell carcinoma (SCC) of the oral tongue is one of the most common malignant lesions in the oral cavity and is characterized by presenting a locally invasive and aggressive behavior. The exosomes are responsible for cell-cell communication and may influence tumor progression, metastasis and therapeutic efficacy. Among the cells that can secrete exosomes are tumor cells and immune cells. It is known that the presence of immune cells is important to eradicate tumors. However, recent findings suggest that inflammation may promote tumor growth. The tumor associated macrophages (TAMs) are known to have subtypes, M1 and M2, that secrete exosomes. This study?s goal was to observe the behavior of derivatives TAMs exosomes, subtypes 1 and 2, against human cell culture SCC-25, HSC-3 and SAS derived from SCC of oral tongue, through the analysis of invasiveness, proliferation and viability of tumor cells in the presence of exosomes. It was observed that the microvesicles derived from TAMs are positive for CD63, characterizing them as exosomes. The exosomes of the M2 subtype TAMs were the only ones to present TGF-? marking, as compared with M1 exosomes, THP1 and SCC cell lines, suggesting that M2 exosomes may be responsible for TGF-? expression in tumor cells. In the migration tests, it was found that the SCC-25 cells in the presence of culture medium DMEM F / 12 showed higher invasion capacity in the presence of M2 exosomes (p?0,001) to a concentration of 0.1 ?g/ml. For HSC-3 and SAS cells, there was no significant statistical relationship between the presence of exosomes and invasiveness (p> 0.05) for the exosomes derived from TAMs. When the exosomes were placed in the lower compartment of the transwell, the HSC-3 cells in the presence of M2 exosomes (1.0 ?g/ml) had higher invasiveness (p?0,001). The viability test showed that HSC-3 cells became more viable in the presence of M2 exosomes (p?0.001) at a concentration of 50 ?g/ml. For SCC-25 cells, the result was the same (p?0.05). Immunofluorescence showed the internalization of exosomes in the cell lines studied. These findings suggest that the presence of M2 exosomes in the SCC of the oral tongue cell cultures may be an important research field for future studies of targeted therapies.

Le radiomarquage des macro-biomolécules au fluor-18 représente un défi majeur en radiochimie vu leur importance en imagerie moléculaire. Les macro-biomolécules et en particulier les peptides offrent une diversité moléculaire et un ciblage in vivo souvent plus spécifiques et sélectifs que les molécules de plus faibles poids moléculaires. Cependant, les conditions standards de radiomarquage au fluor-18 seraient destructrices pour de tels composés et ne peuvent être utilisées directement. Peu de méthodes directes de radiomarquage existent et présentent certains inconvénients (température encore élevée, activité molaire faible, méthodes peu versatiles…). C’est pourquoi, le radiomarquage par approche prosthétique en deux étapes reste une méthode de choix. Cette stratégie séquentielle implique tout d’abord la préparation d’une molécule radiofluorée, appelée groupe prosthétique, puis sa conjugaison à la macro-biomolécule dans des conditions chimiques biocompatibles. L’objectif de ce travail de thèse a consisté à développer de nouvelles méthodes générales de radiomarquage de macro-biomolécules visant in fine des applications de radiomarquage direct in vivo. Les enjeux principaux ont été la diminution du temps de marquage pour améliorer les procédés de radiosynthèse compte tenu de la demi-vie du fluor-18 (109,8 min), le besoin d’automatisation ainsi que la vitesse et la bioorthogonalité des réactions de conjugaison pour des applications en milieu complexe et dilué. Tout d’abord, l’étude de trois réactions de chimie click, CuAAC, SPAAC et SPSAC, de vitesse et de biocompatibilité croissantes a été considérée. Le développement de groupes prosthétiques spécifiques de chacune d’entre elles ainsi que leur conjugaison sur des composés modèles ont ensuite été étudiés. Par la suite, une étude méthodologique sur la radiofluoration de pyridines substituées a été initiée afin d’obtenir des entités radiomarquables dans des conditions suffisamment douces permettant la « pré-conjuguaison » à la macro-biomolécule avant l’étape de radiofluoration. Enfin, l’utilisation d’un étiquetage enzymatique, le SNAP-tag, a également été explorée et un substrat radiofluoré spécifique synthétisé. Ces différentes approches ont permis d’élargir le panel des méthodes permettant un radiomarquage efficace et biocompatible de macro-biomolécules
Fluorine-18 radiolabeling of biologics is a challenge in radiochemistry due to their increasing interest in molecular imaging. Biologics, and particularly peptides, offer molecular diversity and often higher specific and selective in vivo targeting compared to low molecular weight molecules. However, fluorine-18-radiolabeling standard conditions could not be used directly because biologics would suffer from these drastic conditions. Only a few direct radiolabeling methods are available and present some drawbacks (high temperature, low molar activity, lack of versatility…). Therefore, a two-steps prosthetic radiolabeling approach remains the method of choice. This sequential strategy involves the preparation of a fluorine-18-labeled molecule, called prosthetic group, which is then conjugated with the macromolecule in biocompatible chemical conditions. The aim of this PhD work was to develop new general methods for the radiolabeling of biologics directed toward in vivo radiolabeling applications. Major issues were time and radiosynthesis processes with regard to the fluorine-18 half life, automatization as well as constant rate and bioorthogonality of conjugation reactions for applications in complex and diluted environment.The first part is devoted to the study of three click chemistry reactions, CuAAC, SPAAC and SPSAC leading to enhanced reaction rates and biocompatibility. Specific prosthetic groups were developed for each reaction and their conjugation was studied with model compounds. Furthermore, a methodological study involving the radiofluorination of substituted pyridines was initiated for the production of entities for mild radiolabeling conditions compatible with a “pre-conjugation” to the biologics before radiofluorination. Finally, an enzymatic labeling approach using the SNAP-tag self-labeling enzyme was explored and a specific radiofluorinated substrate was synthesized. These different approaches allowed an extent of the panel of methods for effective and biocompatible radiolabeling of biologics

Flavonoids are a class of secondary metabolites, the majority of which are present in glucosylated form. Glucosyltransferases catalyze glucosylation by transferring glucose from UDP-activated sugar donor to the acceptor substrates. This research is focused on the study of the effect of a single point mutation on enzyme activity, characterization of a flavonol specific 3-O-glucosyltransferase (Cp-3-O-GT) mutant- P145T, and further modification of the clone to cleave off tags from recombinant wild type and P145T mutant proteins in order to crystallize the proteins. Multiple sequence alignment and homology modeling was done to identify candidate residues for mutation. Cp-3-O-GT was modeled with a flavonoid 3-O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GTP145T mutant to test the hypothesis that that mutation of proline by threonine in Cp-3-O-GT could alter substrate or regiospecificity of Cp-3-O-GT. While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to flavonols. This is significant because flavanones and flavones do not contain a 3-OH group. HPLC was performed to identify the reaction products. Early results indicated that the mutant protein glucosylates naringenin at the 7-OH position forming prunin. Results are being used to revisit and refine the structure model. In other related work, a thrombin cleavage site was inserted into wild type and recombinant P145Tenzyme and we are currently working on transformation into yeast for recombinant protein expression. Cleaving off tags is a pre-requisite to future efforts to crystallize the proteins. Solving the crustal structures will make a significant contribution to the structural and functional study of plant flavonoid GTs in general and Cp-3- O-GT in particular.

A produção de aves no Brasil vem crescendo na ordem de 10% a cada ano, o que se explica pela atualização constante da tecnologia do setor (http://www.abef.com.br). Sendo a carne de frango a fonte de proteína animal mais barata e acessível ao consumidor, há necessidade de se produzir cada vez mais animais com maior acúmulo de massa muscular. Para isso, o entendimento dos mecanismos celulares e moleculares envolvidos na formação da musculatura esquelética é de extrema relevância. Os fatores miogênicos, genes responsáveis pela determinação e diferenciação de células musculares, foram clonados e progressos significativos foram desenvolvidos quanto ao controle da expressão dos mesmos. A utilização da técnica de seqüenciamento de DNA possibilita a identificação e caracterização de novos genes envolvidos na complexa rede de fatores que regulam a formação da musculatura esquelética em aves. Neste estudo, foram construídas duas bibliotecas de cDNA (fase embrionária e pós-eclosão) de músculo peitoral de uma linhagem de corte (TT) e uma biblioteca da fase embrionária de uma linhagem de postura (CC). A análise das seqüências EST (Expressed Sequence Tags) foi utilizada para identificar possíveis novos genes envolvidos no processo de formação da musculatura esquelética. As seqüências EST identificadas possibilitaram a construção de um banco com 6247 ESTs da musculatura peitoral das linhagens de corte e postura nas duas fases de desenvolvimento. Com o intuito de estabelecer uma relação entre o perfil de expressão dos fatores miogênicos: MyoD, MRF4 e miogenina; e dos genes Pax-3 e miostatina e a formação e maturação das fibras musculares, foi utilizada a técnica de PCR em tempo real. Em geral, a expressão dos fatores miogênicos foi maior na linhagem de corte em relação à de postura nas idades estudadas. Este estudo deverá contribuir para as áreas celular e molecular de desenvolvimento, além de fornecer recursos úteis aos programas de melhoramento genético de aves que visam obter animais com maior acúmulo de massa muscular.
Brazilian’s chicken production is increasing annually around 10%, which can be explained by the current technology applied to this sector (http://www.abef.com.br). Being chicken’s meat the cheapest animal protein source for consumers, there is a need to produce even more animals with increased muscular mass. For this purpose, understanding the molecular and cellular mechanisms involved with the skeletal muscle development is of great relevance. The myogenic factors, genes responsible for the determination and differentiation of muscle cells, were cloned and significant progress was made on the control of their expression. The use of DNA sequencing technique allows the identification and characterization of new genes involved in the complex chain of factors signalling systems that regulates the expression of avian skeletal muscles. In this study, two cDNA libraries (embryonic and post-hatching phases) were constructed from the breast muscle of a chicken broiler line (TT) and one library, from the embryonic phase, from a chicken layer line (CC). The EST (Expressed Sequencing Tags) analysis was used to identify probable new genes involved in the skeletal muscle development. The identified ESTs were used to generate a database containing 6247 breast muscle ESTs from two chicken lines in two development phases. Real time PCR was employed with the aim of establishing a relationship among the expression profile of myogenic factors (MyoD, MFR4, and myogenin), Pax-3 and myostatin genes with the formation and maturation of muscle fibers. In general, the expression of myogenic factors was greater in the broiler than in the layer chicken line in the phases under study. These results should contribute to other cellular and molecular development studies besides providing useful resources for chicken breeding programs whose objective is the deposition of skeletal muscle mass.

La biofiltración ha demostrado ser una tecnología exitosa para la eliminación del H2S y del NH3 de las corrientes de gases contaminadas. El tema asume relevancia pues se establece como tecnología limpia y sostenible. Debido a su organización los biofiltros son considerados ecosistemas artificiales complejos en donde las variables ambientales y la composición de la comunidad están estrictamente relacionadas y de ellas depende el éxito del proceso. La diversidad de bacterias y la dinámica de la comunidad son elementos importantes del componente biológico aunque su conocimiento es aún muy limitado. Para comprenderlo mejor se necesitan perfiles poblacionales elaborados mediante herramientas de biología molecular tales como la pirosecuenciación e hibridación con fluorescencia in situ (FISH). Este fue el eje temático de la presente tesis: Estudiar la composición y la dinámica de comunidades de Eubacteria de biofiltros percoladores mediante pirosecuenciación 454-Roche (tag-454). También se procedió a optimización de protocolos FISH. Para ambas herramientas se evaluó la cobertura de la diversidad y su efectividad para el estudio y seguimiento del componente biológico.Se estudiaron dos sistemas de desulfuración de biogás, uno aerobio y otro anóxico, y un tercer sistema de tratamiento de corrientes ricas en NH3. Los resultados obtenidos demostraron que la comunidad fue capaz de sostenerse bajo las condiciones de operación probadas. En el biofiltro de desulfuración aerobia ocurrió un cambio drástico en la composición de la microbiota en función de la acidificación del medio (pH; 7-2.5), y que la pérdida de diversidad fue compensada por la eficacia de las poblaciones acidófilas. En el biofiltro anóxico los cambios poblacionales no afectaron, en general, en el rendimiento de la desulfuración del biogas, y la actividad de Sedimenticola fue determinante para el éxito del sistema. En el biofiltro percolador de NH3 se comprobó los efectos combinados de tiempos de residencia con diferentes concentraciones de entrada del gas. Las alteraciones en dichos parámetros produjeron cambios significativos en la comunidad nitrificante. Las condiciones favorecieron el crecimiento de Comamonas, Nitrosomonas (AOB) y Nitrobacter (NOB). Fue interesante encontrar una presencia marcada de bacterias desnitrificantes, cosa que no perjudicó al rendimiento de la nitrificación. En los estudios con tag-454 se encontró dificultad a la hora de asignar identidad a secuencias, lo que se justificó por la longitud insuficiente de los fragmentos y por la falta de cobertura de las bases de datos. De todos modos los resultados indicaron que la diversidad fue representada adecuadamente, permitiendo una cobertura muy buena de las distintas comunidades hecho que corrobora la efectividad de la aproximación. El trabajo de optimización de la FISH se enmarca en el conocimiento previo de la diversidad microbiológica a la hora de elegir sondas y se comprobó la influencia de la autofluorescencia del S0 sobre el recuento de células. Los resultados mostraron que la elección de los fluorocromos fue fundamental para eliminar la autofluorescencia de las partículas de S0 que originaban datos sobre-estimados de abundancia relativa.
Biofiltration has proved to be a successful technology for removal of H2S and NH3 from the contaminated gases. The subject assumes significance as it is established as a clean and sustainable technology. Due to its organization biofilters are considered complex artificial ecosystems, where environmental variables and community composition are strictly related and on them depends the success of the process. The bacterial diversity and community dynamics are very important elements of the biological component, but their knowledge is still very limited. To better understand it, populational profiles elaborated by tools such as molecular biology and genomic DNA pyrosequencing of fluorescence in situ hybridization (FISH) are required. This was the theme of this thesis: To study the composition and dynamics of Eubacteria communities in three trickling biofilters using the 454-Roche (tag-454). FISH protocols were also optimized. Two systems of biogas desulphurization were studied, one aerobic and one anoxic, plus a third system for treatment of currents rich in NH3 . The acquired results showed the ability of the community to sustain itself under the tested operational conditions. In the aerobic biofilter desulfurization it occurred a drastic change in the composition of the microbiota in terms of environmental acidification; (pH; 7-2.5), and that biodiversity loss was offset by the effectiveness of acidophilic populations. In the anoxic biofilter, changes did not affect the population in general, the performance of biogas desulfurization and Sedimenticola activity was crucial to the success of the anoxic desulfurization. In the NH3 trickling biofilter, it was proved the combined effects of residence time and different concentrations of gas entrance. Alterations in said parameters produced meaningful changes in the nitrifying community. The resulting conditions favored the growth of the Comamonas, Nitrosomonas (AOB) and Nitrobacter (NOB). It was interesting to find a strong presence of denitrifying bacteria, which did not affect the nitrification performance. In studies using tag-454, the difficulty of assigning identity to the readings of some of the found sequences, which was justified by the insufficient length of the fragments and the lack of databases coverage. Regardless, the results indicated that the diversity represented the 95% of similarity, and in the end there was a very good coverage of different communities. The relative abundance data allowed the explanation of the dynamics of said communities, fact that corroborates the approximation’s efficiency. The optimization of work by FISH relays in prior knowledge of microbial diversity when choosing probes, and proved the influence of the S0 autofluorescence over the cell count. The results showed that the choice of fluorochromes was fundamental to eliminate the autofluorescence of S0 particles that originated over-estimated data of relative abundance.

L'océan est le plus grand réservoir d'énergie solaire de notre planète. La quantité de chaleur qu'il est capable de stocker est modulée par sa circulation complexe, opérant sur une vaste gamme d’échelles allant du centimètre à la dizaine de milliers de kilomètres. Cette thèse s'intéresse à deux types de processus océaniques: les tourbillons de mésoéchelle, d'une taille de 100 à 300 km, et les fronts de sous-mésoéchelle, d'une taille inférieure à 50 km. L'idée communément admise est que les mouvements agéostrophiques de sous-mésoéchelle sont principalement confinés à la couche de mélange océanique de surface et sont faibles dans l'océan intérieur. Cette vision classique de la dynamique océanique repose sur l'hypothèse que l'océan intérieur est en équilibre quasi-géostrophique, empêchant la formation de forts gradients de densité en profondeur. Cette thèse remet en question ce paradigme en se basant sur des observations CTD in situ à haute résolution collectées par des éléphants de mer instrumentés, des images satellite d’élévation de la surface de l’océan, et des sorties de modèle à haute résolution dans le Courant Circumpolaire Antarctique.Les résultats indiquent que les mouvements agéostrophiques sont (i) générés par le champ tourbillonnaire de mésoéchelle via des processus defrontogenèse, et (ii) ne sont pas limités à la couche de mélange de surface ; bien au contraire, ils pénètrent dans l'océan intérieur jusqu'à 1000 m deprofondeur. Ces fronts agéostrophiques de sous-mésoéchelle sont associés à d'importants flux de chaleur dirigés de l'intérieur de l'océan vers la surface, d'une amplitude comparable aux flux air-mer.Cet effet peut potentiellement altérer la capacité de stockage de chaleur de l'océan et devrait être le plus fort dans les zones tourbillonnaires telles que le Courant Circumpolaire Antarctique, le Kuroshio et le Gulf Stream, les trois courants clefs du système climatique. Il apparaît ainsi que les fronts agéostrophiques de sous-mésoéchelle représentent une voie importante, mais encore largement méconnue, pour le transport de chaleur, de nutriments et de gaz entre l'intérieur et la surface de l'océan, avec des répercussions potentiellement majeures pour les systèmes biogéochimique et climatique
The ocean is the largest solar energy collector on Earth. The amount of heat it can store is modulated by its complex circulation, which spans a broad range of spatial scales, from centimeters to thousands of kilometers. This dissertation investigates two types of physical processes: mesoscale eddies (100-300 km size) and submesoscale fronts (£ 50 km size). To date, ageostrophic submesoscale motions are thought to be mainly trapped within the ocean surface mixed layer, and to be weak in the ocean interior. This is because, in the classical paradigm, motions below the mixed layer are broadly assumed to be in quasigeostrophic balance, preventing the formation of strong buoyancy gradients at depth. This dissertation introduces a paradigm shift; based on a combination of high-resolution in situ CTD data collected by instrumented elephant seals, satellite observations of sea surface height, and high-resolution model outputs in the Antarctic Circumpolar Current, we show that ageostrophic motions (i) are generated by the backgound mesoscale eddy field via frontogenesis processes, and (ii) are not solely confined to the ocean surface mixed layer but, rather, can extend in the ocean interior down to depths of 1 000 m. Deepreaching ageostrophic fronts are shown to drive an anomalous upward heat transport from the ocean interior back to the surface that is larger than other contributions to vertical heat transport and of comparable magnitude to air-sea fluxes. This effect can potentially alter oceanic heat uptake and will be strongest in eddy-rich regions such as the Antarctic Circumpolar Current, the Kuroshio Extension, and the Gulf Stream, all of which are key players in the climate system. As such, ageostrophic fronts at submesoscale provide an important, yet unexplored, pathway for the transport of heat, chemical and biological tracers, between the ocean interior and the surface, with potential major implications for the biogeochemical and climate systems

The ability to express and purify soluble protein in significant amounts is a prerequisite for the structure analysis of biological macromolecules by spectroscopic techniques. Nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopies are powerful biophysical techniques which are widely used in structural biology. This thesis focuses on the use of different fusion constructs and tagging strategies to produce samples for subsequent NMR and EPR measurements. Following the general introduction of Chapter 1, Chapter 2 explores N-terminal fusions based on the nucleotide sequence of the T7 gene 10 which translates into the hexapeptide MASMTG. A systematic comparison of the expression levels with and without MASMTG tag was conducted for five different proteins (E. coli aspartate/glutamate-binding protein (GBP), green fluorescent protein (GFP), MutT Homolog 1 (MTH1), dengue virus type 2 NS2B-NS3 protease (DENp) and Methanosarcina barkeri pyrrolysyl-tRNA synthetase (nbCRS)) in both a cell-free protein synthesis setup and in vivo in E. coli. The expression yields of DENp, GFP and nbCRS were greatly enhanced by the MASMTG tag, barely changed for GBP and decreased for MTH1. This result shows that the N-terminal fusion with a tag from a protein known to express in very high yields can indeed enhance the expression yields for some proteins even if they are already codonoptimized for expression in E. coli. Chapter 3 describes the development of an efficient and inexpensive strategy for site-specific paramagnetic tagging of oligonucleotides, which allowed measurements of pseudocontact shifts (PCS) in the DNA using lanthanide ion tags. The strategy relies on commercially available oligonucleotides synthesized with a phosphorothioate group. HPLC conditions were developed to separate the two phosphorothioate diastereomers and their configurations determined by an enzymatic assay with snake venom phosphodiesterase. The new lanthanide-binding tag C10 was attached by alkylating the phosphorothioate group. PCS measurements were carried out following hybridization with the complementary DNA strand to form DNA duplexes. Although the PCSs were relatively small, they confirmed the site-specific attachment of the tag. Larger PCSs were observed for the SP than the RP diastereomer and good correlations were observed between backcalculated and experimental PCSs, in particular for the SP-phosphorothioate oligonucleotide, indicating that this tagging approach delivers reliable long-range structural information. Chapter 4 describes the preparation of a homeodomain-DNA complex with three different types of spin labels for double electron–electron resonance (DEER) measurements. A Gd3+ tag was introduced into the homeodomain by copper-catalyzed click reaction with a genetically encoded unnatural amino acid (p-azidophenylalanine) and an EDTA-Mn2+ tag was introduced by reaction with Cys39. With a nitroxide tag attached to a phosphorothioate group in the DNA, DEER measurements determined the distances between the three labels in the triple-tagged homeodomain-DNA complex. The experimentally determined Mn2+−nitroxide and Gd3+−Mn2+ distance distributions agreed well with the distances predicted from the NMR structure of the complex, whereas the calculated Gd3+−nitroxide distance was ∼0.5 nm longer than the experimental one. This study demonstrated the potential of three different spin labels to obtain three independent distance restraints in a single sample. Chapter 5 describes experiments for the site-specific incorporation of the unnatural amino acids Boc-lysine and TMS-lysine into proteins using the Methanosarcina mazei pyrrolysine-tRNA synthetase (PylRS) mutants Y384F/Y306A and Y384F/Y306G/I405R in E. coli BL21 (DE3) and E. coli B95-DA. While Boc-lysine could readily be incorporated, the experiments to incorporate TMS-lysine were unsuccessful. Chapter 6 describes strategies explored to produce uniformly 15N-labelled MARCKS peptide.In vivo expression of this peptide had proven notoriously difficult but was successfully achieved by fusion with a trigger-factor–ubiquitin (TF-Ub) construct, which can be cleaved with a ubiquitinase to release the free peptide. 15N-HSQC spectra were recorded of the peptide in complex with the N60D mutant of calmodulin (CaM) loaded with calcium, and chemical shift changes and paramagnetic relaxation enhancements (PRE) detected in the presence of paramagnetic lanthanide ions confirmed specific binding to CaM and interactions with the N-terminal domain of CaM. This establishes the basis for future structural analysis of the binding mode of the MARCKS peptide to CaM. An intein strategy was unsuccessful for the expression of the MARCKS peptide, but the system successfully produced tag-free PylRS and allowed its purification in soluble form. The purified PylRS was inactive in the in-house cell-free protein synthesis (CFPS) system, indicating that the well-known problems with the activity of this enzyme are not associated with the presence of commonly used purification tags.

The dicyemid mesozoans (Dicyemida Van Beneden 1882) are a poorly-understood group of marine organisms that are found with high intensities in the renal appendages of benthic cephalopods. The majority of the research focusing on this group is from the northern hemisphere, with very few studies examining the dicyemid fauna of southern hemisphere cephalopod species. Confusion also exists in the literature on the validity of certain families, genera and species within this phylum, and the phylogenetic framework for the dicyemids is scarce. The few studies that have examined dicyemid molecular genetics focus only on single taxon or sole aspects of genome organisation. Furthermore, key parts of the life cycle of dicyemid parasites are unresolved and their position in the Tree of Life is uncertain. My thesis highlights the taxonomic confusion in the literature that surrounds the Dicyemida, and presents a comprehensive list of all dicyemid species currently described to date (Chapter 2). Ten cephalopods species from Australian waters were collected and examined for dicyemids parasites, resulting in new dicyemid species descriptions (Chapters 3, 4 and 5). Host eggs and filtered seawater samples were collected from the cuttlefish mass breeding aggregation at Upper Spencer Gulf, South Australia, Australia, to assess the unknown host life cycle stage where new infection by the dispersive dicyemid embryo occurs. No dicyemid DNA was detected in any host egg or environmental samples, suggesting new infection occurs after the host embryo hatches rather than at the egg stage (Chapter 6). Patterns of infections, prevalence, species richness, co-infection and co-occurrence of dicyemids among infected cephalopods species were explored (Chapter 7). Host size in general did not influence patterns of infection, however where dicyemid species co-occurred, restriction to discrete host sizes was observed, suggesting competition between species may be an important factor leading to niche separation. Calotte shape was found to vary between dicyemid species that co-occurred within a single host individual. Additionally, dicyemid fauna composition was found to vary with host geographical collection locality, alluding to the potential use of dicyemid parasites as biological tags (Chapter 7). The complete cytochrome c oxidase subunit I (COI) minicircle molecule, including the COI gene plus a non-coding region, was sequenced from nine dicyemid species, and comparisons in sequence composition and size were made between and within species (Chapter 8). The first phylogeny of dicyemids including multiple taxa from the two genera that combined contain over 90% of the nominal described species was estimated from Bayesian inference and maximum likelihood analyses. Monotypic species clades were observed, however the paraphyly to the genera suggests classification based on morphological traits may need revision (Chapter 8). The hypothesis that parasite genetics of infected cephalopods will allow for a deeper insight into population structuring compared to that gained with complementary methods was tested, with dicyemid mesozoans infecting giant Australian cuttlefish (Sepia apama) as the chosen system (Chapter 9). The population structure of S. apama previously inferred from host morphology, behaviour and genetics was supported from dicyemid parasite mitochondrial haplotype phylogeography, with an analysis of molecular variance (AMOVA) providing an alternative insight into structuring of this cuttlefish species. This result suggests that in the future, a holistic approach that incorporates parasite and host data (morphology and genetics) should be used to assess cephalopod population boundaries. An invited review article on the use of parasites as biological tags to assess the population structure of marine organisms is presented as the final data chapter (Chapter 10). Comments are made on the guidelines for selecting a parasite species as a reliable tag candidate, the need to incorporate parasite genetic information and the benefits of a multidisciplinary approach. The direct outcomes of my study include the description of the first dicyemid species from Australian waters, insights into the unknowns in the dicyemid life cycle, presentation of the first dicyemid phylogeny allowing taxa classification to be assessed outside of the sole morphological approach and analysis of the use of dicyemid parasites as biological tags, supporting the integration of dicyemid parasite genetics alongside other complementary methods to assess cephalopod population structure. In summary, my study has significantly contributed to the field of dicyemid research, increasing both fundamental and applied knowledge on this enigmatic group of organisms.
Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2014

In order to avoid, reduce and offset the impact that human activities are having on the health of the world's oceans, the international demand for a successful ecosystem-based approach to the marine environment is growing in popularity. Major European maritime policies and other international agencies advocate that, in order to effectively protect the marine environment, there is a need to establish ecologically coherent networks of marine protected areas (MPAs). Achieving ecological coherence is, however, a complex task given the magnitude of the concept and the lack of clear operational definitions. Therefore, assessing ecological coherence requires multiple approaches to analyse not only the location, size and spacing of protected sites already selected for protection, but also infer about new sites to enhance network effects. The aim of this thesis is to develop and use different tools to assist, support and help to promote the assessment of the ecological coherence of networks of MPAs, with a focus on continental Portugal. Three complementary methods were applied using the mussel Mytilus galloprovincialis as a model species to estimate connectivity via larval dispersal along the Portuguese west coast. The first study described the use of natural geochemical tags to trace back natal origins of mussels and describe direct measures of demographic connectivity patterns (dispersal trajectories and distances). It focused in the Arrábida and Berlengas Marine Protected Areas (MPAs) and emphasized the significance of source and sink populations and self-recruitment within MPAs. The second study resulted from the need to quantify mussel bed coverage in order to apply the connectivity estimates into broader scale models of larval dispersal. The use of low altitude and high-resolution drone imagery, 3D surface models and ground-based observations proved to be a viable and powerful tool for surveying intertidal ecosystems at an ecological relevant spatial scale. The effect of environmental variables (substrate complexity and wave exposure) on mussel density and size was also investigated. Maximum mussel density occurred at intermediate values of wave exposure in winter while smaller sized mussels prevailed at high values of wave exposure. The combination of the empirical connectivity estimates, with mussel demographic and biological parameters (reproductive output, larval behaviour, mortality) allowed the validation of a broad scale numerical biophysical model in the third study This model combined oceanographic and demographic information with larval biology, and was used to simulate dispersal of M. galloprovincialis larvae along the Western Iberian coast. The biophysical model simulated and accounted for uncertainty of the geochemical method in the assignment of source populations and resulted in unprecedented levels of correspondence among independent connectivity estimates at small spatial scales. This fine-tuned model can be used in the future to investigate metapopulation dynamics with applications in MPA management strategies. Finally, a quantitative-based ecological approach was used to value biodiversity along the Portuguese continental shelf. The marine biological valuation protocol summarized and combined existing biological and ecological spatial information of different ecosystem components into an overall map of marine biological value, highlighting hotspots of ecological significance. This study also evaluated the extent to which high biological value was contained in the current and projected Natura 2000 sites in Portugal. By integrating the information of marine connectivity pathways, with a broad biological assessment of the Portuguese coast, this thesis seeks to build upon existing MPA assessment criterion and take a step forward in making it practical and applicable to the Portuguese setting.
A pressão internacional para encontrar abordagens ecossistémicas eficazes para evitar, reduzir e compensar o impacto das atividades humanas na saúde global dos oceanos, está a aumentar. Nesse sentido, as principais políticas marítimas europeias e de outras agências internacionais, defendem a necessidade de estabelecer redes ecologicamente coerentes de áreas marinhas protegidas (AMPs). Atingir a coerência ecológica é, no entanto, uma tarefa complexa dada a magnitude do conceito e a falta de definições operacionais objectivas. Deste modo, avaliar a coerência ecológica requer abordagens múltiplas para analisar a localização, tamanho e espaçamento entre áreas protegidas já selecionadas, mas também para inferir sobre a designação de novos locais e melhor a performance da rede. O objetivo desta tese é desenvolver e utilizar diferentes ferramentas de forma a apoiar e promover uma avaliação da coerência ecológica da rede de AMPs em Portugal continental. Este trabalho utilizou três métodos complementares para estimar conectividade larvar ao longo da costa oeste portuguesa, usando o mexilhão Mytilus galloprovincialis como espécie-modelo. O primeiro método retrata o uso de etiquetas geoquímicas naturais para fazer o rastreo da origem natal de mexilhões, quantificando padrões de conectividade demográfica (trajetórias de dispersão e distâncias). Concentrou-se nas AMPs da Arrábida e Berlengas, salientando a importância das populações que servem como fonte de indivíduos para dispersão e auto-recrutamento dentro das AMPs. O segundo método resultou da necessidade de quantificar a cobertura de mexilhão no intermareal rochoso, para futura aplicação em modelos de dispersão larvar de larga escala. O uso de imagens aéreas de baixa altitude e de alta resolução, modelos de superfície tridimensionais e de observações in situ, provaram ser uma ferramenta viável para monitorizar ecossistemas intermareais numa escala espacial ecologicamente relevante. O efeito de variáveis ambientais (complexidade do substrato e exposição às ondas) na densidade e tamanho do mexilhão foi também investigado. A densidade máxima de mexilhão no substrato rochoso ocorreu com valores intermédios de exposição às ondas no Inverno, enquanto que altos valores de exposição às ondas resultaram num menor tamanho dos mexilhões. No terceiro estudo, a combinação das estimativas empíricas de conectividade, com parâmetros demográficos (fertilidade, comportamento larval, mortalidade) permitiram a validação de um modelo numérico biofísico de larga escala. Este modelo integrou informações oceanográficas, demográficas e de biologia larvar, para simular a dispersão de larvas de M. galloprovincialis ao longo da costa oeste da Peninsula Ibérica. O modelo incorporou e simulou também a incerteza associada à atribuição da origem das populações referente ao método geoquímico. Os resultados demonstraram altos níveis de correspondência entre as estimativas independentes de conectividade a uma pequena escala espacial. Este modelo validado pode ser usado no futuro para investigar dinâmicas metapopulacionais com aplicações em estratégias de gestão de AMPs. Finalmente, utilizou-se uma abordagem ecológica para valorizar a biodiversidade marinha ao longo da plataforma continental portuguesa. Este protocolo de avaliação biológica integrou informações biológicas e ecológicas espaciais, para diversos componentes do ecossistema. Os resultados foram ilustrados num mapa global de valor biológico marinho, destacando hotspots de significância ecológica. Este estudo avaliou também em que medida as zonas de alto valor biológico estavam incluido nas áreas protegidas da rede Natura 2000 em Portugal (actuais e propostas). Ao integrar a informação das vias de conectividade marinha, com uma ampla avaliação biológica da costa portuguesa, esta tese procura contribuir para os critérios de avaliação de redes de AMPs e dar um passo em frente no sentido de o tornar aplicável ao panorama português.
De internationale vraag naar een succesvolle ecosysteem-gebaseerde aanpak om de impact van menselijke activiteiten op de gezondheid van de oceanen te vermijden, te reduceren of te compenseren, neemt toe in populariteit. Belangrijke Europese maritieme beleidsinstanties en andere internationale agentschappen benadrukken dat het oprichten van ecologisch coherente netwerken van beschermde zeegebieden noodzakelijk is om het mariene milieu doeltreffend te beschermen. Echter, het bereiken van ecologische coherentie is een complexe taak gezien het ruime concept en het ontbreken van eenduidige operationele definities. Het vaststellen van ecologische coherentie vereist dus verschillende benaderingen. Op die manier kan niet alleen de locatie, de grootte en afstand van reeds geselecteerde gebieden voor bescherming bepaald worden, maar ook nieuwe locaties die het netwerkeffect versterken. Het doel van deze thesis is het ontwikkelen en toepassen van verschillende methodes als hulpmiddel om de ecologische samenhang van beschermde zeegebieden te promoten, en dit met focus op het Portugese continentale plat. Drie complementaire methodes werden toegepast om de connectiviteit via larvale dispersie in te schatten langs de Portugese westkust en dit telkens toegepast op de mossel Mytilus galloprovincialis als modelorganisme. De eerste studie beschrijft het gebruik van natuurlijk voorkomende geochemische tracers om de oorsprong van de mossels te achterhalen en documenteert dus de demografische connectiviteitspatronen (verspreidingsroutes en afstanden). De focus lag op de beschermde zeegebieden van Arrábida en Berlengas, en benadrukte het belang van ‘source–and–sink’ populaties en zelf-recrutering binnen de beschermde gebieden. In een tweede studie werd de oppervlakte van mosselbedden gekwantificeerd zodat de connectiviteitsschattingen konden toegepast worden op modellen van larvale dispersie op grotere schaal. Het gebruik van beeldmateriaal met hoge resolutie verzameld door drones op lage hoogte, 3D-oppervlakte modellen en veldobservaties bleek een efficiënte methode om intertidale ecosystemen te bestuderen op een ecologisch relevante schaal. De combinatie van empirische connectiviteitsschattingen en demografische en biologische parameters (oppervlakte van mosselbedden, gedrag van larves, mortaliteit, reproductieve output en timing) laat de validatie toe van een grootschalig numeriek, biofysisch model. Het effect van de omgevingsvariablen (zoals substratum complexiteit en blootstelling aan golven) op de mosseldichtheid en -grootte werd ook onderzocht. Maximum mosseldichtheid kwam voor bij intermediaire waarden van golfblootstelling in de winter en kleinere mosselen domineerden bij hoge waarden van blootstelling aan golven. Dit model combineert oceanografische en demografische informatie met larvale biologie, en werd gebruikt om de dispersie van M. galloprovincialis larven langs de West-Iberische kust te simuleren. Het biofysisch model hield rekening met de onzekerheid van de geochemische methode in het toekennen van de ‘source’ populaties en resulteerde in ongeziene overeenkomsten tussen de onafhankelijke connectiviteitsschattingen op kleine schaal. Vanaf nu kan dit verfijnde model gebruikt worden om de dynamieken van metapopulaties te onderzoeken en kan het dus bijdragen aan weldoordachte strategieën met betrekking tot het oprichten van beschermde zeegebieden Tot slot werd een kwantitatief-gebaseerde ecologische aanpak gebruikt om de biodiversiteit te bepalen langs het Portugees continentaal plat. Het protocol voor mariene biologische waardebepaling combineerde bestaande biologische en ecologische informatie van verschillende subzones in een kaart die de biologische waardebepaling weergeeft, en hotspots van ecologisch belang identificeert. Deze studie evalueerde ook de mate waarin grote biologische waarde vervat zit in de huidige en toekomstige Natura 2000 gebieden in Portugal. Door het integreren van informatie over mariene connectiviteitsroutes, met breed biologisch onderzoek langs de Portugese kust, bouwt deze thesis verder op het bestaande criterium voor de selectie van beschermde gebieden, en maakt het praktisch en toepasbaar voor Portugal.
MARES PhD Grant (MARES_12_10); FPA 2011-0016
Programa Doutoral em Ciências do Mar (Mares)

The magnitude of the document collection in the biology domain boosts the demand for effective and efficient literature mining and knowledge discovery that can help biologists to gather and make use of the knowledge encoded in text documents. In this thesis, we present three different pattern-based techniques to target two important tasks of biological information extraction: entity recognition and relation extraction. The first technique is an unsupervised method to automatically extract domain-specific prefix and suffix characters from biological corpora. The extracted characters are integrated into the parametrization of an existing system for biological entity recognition in order to aid the system to annotate biological entities. The second technique is an approach to identify sentences that describe interactions between co-occurring biological entities using patterns defined as a sequence of specialized Part-of-Speech (POS) tags that capture the structure of key sentences in the scientific literature. Each candidate sentence for the classification task is encoded as a POS array and then aligned to a collection of pre-extracted patterns. The quality of the alignment is expressed as a pairwise alignment score. The most innovative component of this work is the use of a Genetic Algorithm (GA) to maximize the classification performance of the alignment scoring scheme. The third technique is a graph matching-based approach to extract complex biological events from the scientific literature. Sentences are represented as dependency graphs, and biological event rules are extracted from sentences as minimal dependency graphs that capture the typical contextual structures of biological events. We investigate whether the subgraph matching problem can be used in the BioNLP field to extract biological events by searching for subgraphs isomorphic to the graphs of event rules within the graphs of sentences.

Tese de mestrado. Biologia (Biologia da Conservação). Universidade de Lisboa, Faculdade de Ciências, 2009
A Branca-Portuguesa é uma espécie de borboleta (Insecta, Lepidoptera) cuja distribuição abarca a região Atlântico-Mediterrânica entre Portugal e Itália, Marrocos e Argélia, em núcleos fragmentados e divergentes do ponto de vista morfológico. O isolamento das suas populações advém fundamentalmente da sua especificidade ecológica na dependência de solos calcários onde se desenvolve um matagal mediterrânico de fácies calcícola e das plantas de que se alimentam as lagartas, as Assembleias. Este estudo, apoiado na importância das borboletas como elementos basilares nos ecossistemas, bem como na importância que a sua promoção representa para a conservação dos seus habitats, tem origem na lacuna de conhecimento existente sobre esta espécie em Portugal. A nível nacional, até ao presente apenas era conhecida uma única população, na Serra da Arrábida. A sinergia entre os dados cartográficos de distribuição dos calcários e das plantas alimentícias permitiu criar um mapa de distribuição potencial da espécie em Portugal e o trabalho de campo comprovou a existência de populações previamente desconhecidas no Alentejo e no Maciço Calcário Estremenho. Todos os locais de ocorrência partilham uma gama de características fundamentais para a sua subsistência. Através do seguimento e caracterização de todas as fases do seu ciclo-de-vida preencheu-se também uma outra lacuna de conhecimento. Através de um marcador molecular mitocondrial pretendeu-se avaliar o grau de diferenciação genética das populações europeias da espécie bem como inferir a sua história filogeográfica. E. tagis apresenta uma elevada diversidade genética não estruturada segundo a taxonomia e terá subsistido na Península durante o Quaternário em isolados policêntricos que sucessivamente entraram em contacto, tornando o padrão filogeográfico difuso. Com todos estes dados foi possível confrontar informação multidisciplinar para a conservação da Branca-Portuguesa, relevando-se a sua importância como espécie “guarda-chuva” e bio-indicadora de qualidade ecológica, podendo ser integrada, a par de outras espécies prioritárias como “espécie bandeira” dos seus habitats.
The Portuguese Dappled White is a butterfly (Insecta, Lepidoptera) whose distribution encompasses fragmented and morphologically distinct populations in the Atlantico-Mediterranean region from Portugal to Italy, Morocco and Algeria. This fragmentation arises fundamentally from its ecological specificity, depending on limestone soils where a lush Mediterranean calcicole scrubland develops, which is also where its caterpillar foodplants, Candytufts, are to be found. This study has its origin in the absolute lack of knowledge regarding this species in Portugal and is supported by the keystone importance of butterflies for ecosystems and of their promotion towards the conservation of their habitats. At the national level, until now only one population was known to thrive, at Serra da Arrábida. The synergy between cartographic data of the distribution of both limestone soils and the butterfly’s foodplants made possible the production of a potential distribution map for the species in Portugal and subsequent fieldwork unveiled the existence of previously unknown populations in Alentejo and the Maciço Calcário Estremenho. An E. tagis site holds a number of fundamental characters which allow its survival. Through the rearing and description of its early-stages, a knowledge gap was filled-in. With the use of a molecular mitochondrial marker it was possible to evaluate the degree of genetic divergence among the European populations of E. tagis and infer its phylogeography. This species exhibits a high genetic diversity, albeit not structured according to current taxonomy and probably has thrived in the Península during the entire Quaternary in polycentric isolates with subsequent migration between them making the general phylogeographical pattern diffuse. With these data we could confront multidisciplinary information towards the conservation of the Portuguese-Dappled-White, emphasizing its importance as an umbrella species and a bio-indicator of ecological quality. Together with other charismatic, priority species it can also be a good flagship species for its habitats.

Tese de doutoramento em Biologia (Biologia Marinha), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2007
Os linguados Solea solea (Linnaeus, 1758) e Solea senegalensis Kaup, 1858, estão entre os peixes com maior valor comercial, em Portugal. Os indivíduos adultos destas espécies habitam a plataforma continental, enquanto que os juvenis se concentram em áreas costeiras e em particular em estuários. Duas importantes áreas de viveiro para os juvenis destas espécies foram identificadas no estuário do Tejo: a área de Vila Franca de Xira e a de Alcochete. Enquanto que S. solea apenas coloniza a área de viveiro de Vila Franca de Xira, S. senegalensis coloniza ambas as áreas de viveiro. O presente trabalho tem como objectivo aprofundar o conhecimento no que se refere à ecologia de juvenis destas espécies nas áreas de viveiro do estuário do Tejo. O uso do habitat pelos juvenis destas espécies foi analisado em diferentes escalas espaciais, recorrendo a técnicas de análise espacial, análises isotópicas e a um programa de amostragem na zona intertidal que teve em conta os ciclos circum-diário e lunar.Foram desenvolvidos modelos espaciais de qualidade do habitat para ambas as espécies de forma investigar quais as variáveis que definem as áreas onde os juvenis se concentram e que, por isso, deverão ser levadas em conta na elaboração de planos de gestão que visem as populações de linguado ou as áreas estuarinas em questão. A importância da salinidade, temperatura, substrato, profundidade e presença de plataformas vasosas foi confirmada, no entanto, a inclusão da abundância de presas provou ser crucial para a definição das áreas de elevada qualidade ambiental onde as grandes concentrações destas espécies ocorrem de forma consistente. As análises isotópicas aos vários elos das cadeias alimentares de ambas as áreas de viveiro, revelaram que os juvenis de S. senegalensis com idade inferior a 1 ano exibem elevada fidelidade à zona de viveiro onde vivem, enquanto que uma fracção considerável de juvenis com mais de um ano de idade explora ambas as áreas de viveiro. Concluiu-se também, que as cadeias tróficas das duas áreas de viveiro têm uma dependência diferencial das fontes de água-doce. A investigação simultânea do efeito do ciclo circum-diário e lunar sobre uso da zona intertidal por parte dos juvenis de S. senegalensis, permitiu detectar um padrão no qual durante as marés vivas a abundância de juvenis atinge o pico no crepúsculo, enquanto que nas marés mortas o pico é diurno. O maior pico de abundância ocorre no crespúsculo durante a lua-cheia. A análise dos padrões de actividade dos seus principais predadores e informação prévia sobre os efeitos dos ciclos circum-diário e lunar sobre as suas presas indiciam que os padrões de actividade encontrados para S. senegalensis têm uma relação estreita com a dos seus predadores e presas. Foram conduzidas experiências em cativeiro de forma a determinar a influência da temperatura e salinidade sobre a evacuação gástrica de ambas as espécies, assim como para a detecção de alterações no comportamento alimentar devido a pressão predatória. Observou-se que a evacuação gástrica aumenta com a temperatura, para ambas as espécies (com excepção da temperatura experimental de 26ºC, em S. solea). No caso da salinidade, verificou-se um efeito diferente, de acordo com a espécie. A baixas salinidades, S. solea manifestou um aumento da taxa de evacuação gástrica, enquanto que para S. senegalensis registou-se uma diminuição desta taxa. O efeito da temperatura experimental de 26ºC em S. solea foi discutido, concluindo-se que este é possivelmente um indício de que esta espécie se encontra perto do seu limite superior térmico. Este efeito não foi observado em S. senegalensis, provavelmente porque, sendo esta uma espécie com afinidades subtropicais, possuirá um limite superior térmico mais elevado. A diferença detectada nas taxas de evacuação gástrica a 26ºC terá importantes implicações competitivas para S. solea durante os meses mais quentes, quando ambas as espécies de linguado se concentram em águas pouco profundas, ricas em presas, mas onde as temperaturas são consideravelmente mais elevadas do que o óptimo metabólico de S. solea. A experiência de observação comportamental revelou um decréscimo de 10% na actividade de S. senegalensis, quando na presença de um potencial predador. As taxas de evacuação gástrica, juntamente com ciclos de amostragem conduzidos na área de estudo, foram integradas num modelo, de forma a calcular o consumo alimentar de ambas as espécies. O consumo diário de S. senegalensis foi consideravelmente superior ao de S. solea. Os padrões alimentares observados evidenciaram dois picos distintos de actividade alimentar, ao anoitecer e ao amanhecer, estes foram, no entanto, muito mais pronunciados para S. senegalensis do que para S. solea. Uma vez que estudos a latitudes mais elevadas verificaram picos de actividade alimentar em S. solea mais pronunciados e da ordem de magnitude da encontrada no estuário do Tejo para S. senegalensis, concluiu-se ser este mais um indício de que o metabolismo de S. solea está próximo do seu limite térmico, estando, por isso, diminuído. O cálculo do consumo alimentar total, durante os meses de maior abundância destas espécies nas áreas de viveiro, e da disponibilidade alimentar do meio levou à conclusão de que a disponibilidade de alimento não é um factor limitante para os juvenis de linguado do estuário do Tejo. Considerou-se, no entanto, que será necessária mais informação sobre a variabilidade de abundância das comunidades de presas e sobre o consumo por parte de outros predadores para determinar com segurança a capacidade de suporte das áreas de viveiro do estuário do Tejo. A elevada variabilidade inter-anual da abundância de ambas as espécies de linguado deverá, também, ser levada em conta. O estudo do crescimento e condição dos juvenis de S. solea e S. senegalensis focou a variação das taxas de crescimento e da razão RNA-DNA nas sucessivas coortes que colonizam o estuário do Tejo. Focou também, a comparação da qualidade do habitat entre as duas áreas de viveiro e a comparação do crescimento e período de desova num contexto latitudinal. Foram observados padrões de variação do crescimento e da condição que reflectem o processo de colonização estuarina. O crescimento e a condição são mais elevados no início da colonização, diminuindo com o tempo, e a primeira coorte apresenta valores mais elevados que as coortes subsequentes. A comparação das taxas de crescimento calculadas para as duas áreas de viveiro evidenciou valores mais elevados para a área de Alcochete do que para a área de Vila Franca de Xira. A condição geral dos indivíduos de ambas as espécies foi considerada boa, não tendo sido encontrada uma diferença significativa entre as duas áreas. Concluiu-se que, dada a elevada dinâmica característica das áreas de viveiro estuarinas, será necessária a determinação da qualidade ambiental destas áreas durante um período de tempo mais alargado, de forma a investigar a sua variação inter-anual. As taxas de crescimento de S. solea no estuário do Tejo foram superiores às estimadas para latitudes mais elevadas. O uso de ambas as metodologias para monitorização de qualidade ambiental das áreas de viveiro do estuário do Tejo, o cálculo de taxas de crescimento com base nos anéis diários dos otólitos e a determinação da razão RNA-DNA, foi discutido. A determinação da época de desova de S. solea através de contagem retrógrada dos anéis diários dos otólitos e a sua comparação com outras áreas de viveiro ao longo da costa ocidental Europeia, revelou existir uma variação latitudinal na qual a desova é iniciada mais cedo quanto menor a latitude. O impacto de factores climáticos e oceanográficos sobre a imigração larvar para as áreas de viveiro foi investigado. Da análise de uma série temporal descontínua de densidades de juvenis de ambas as espécies (1988-2006) e da sua relação com o índice de oscilação do Atlântico Norte, a prevalência de ventos do quadrante Norte e o caudal do rio Tejo, durante o período larvar, resultou que só para esta última variável foi encontrada uma correlação significativa. A extensão das plumas fluviais na zona costeira será, assim, muito importante, provavelmente devido ao seu papel no transporte de pistas químicas que são utilizadas pelas larvas para direccionar o seu movimento. Isto significa que em anos chuvosos estas pistas químicas atingirão uma área maior aumentando a probabilidade de detecção por parte das larvas. Concluiu-se que a diminuição e maior concentração temporal da precipitação, previstas devido a alterações climáticas, terão um efeito significativo sobre a imigração larvar de ambas as espécies, mas em particular sobre S. senegalensis, uma vez que o seu período de desova é mais alargado. Foi, desta forma, evidenciada a importância da monitorização dos caudais fluviais e do seu efeito sobre a imigração de linguados para áreas de viveiro estuarinas. A estimativa do impacto da pesca sobre estes juvenis, permitiu concluir que esta tem um impacto considerável sobre os efectivos populacionais de ambas as espécies, sendo este, no entanto, mais acentuado para S. solea. Verificou-se que a fracção da população de S. senegalensis afectada por mortalidade por pesca é menor, uma vez que, esta espécie coloniza também a área de Alcochete, onde o esforço de pesca é bastante menor do que em Vila Franca de Xira. Desta forma a área de viveiro de Alcochete funciona como uma zona mais protegida para esta espécie. Foi confirmada a necessidade de revisão da legislação que permite a pesca com arrasto de vara no estuário do Tejo. Foi dado enfâse à importância da integração da informação obtida neste trabalho em planos de gestão dos mana
The soles, Solea solea (Linnaeus, 1758) and Solea senegalensis Kaup, 1858, are among the most valuable commercial fish in Portugal. Two important nursery areas for the juveniles of these species have been identified in the upper area of the Tagus estuary. The present work aimed at investigating the ecology of these species in the Tagus estuary nursery areas. Habitat use was analysed at different spatial scales, using spatial modelling, isotopic analysis and a complex sampling program accounting for the diel and lunar cycles. Prey abundance proved crucial in the prediction of high densities of juveniles. The stable isotope approach revealed low connectivity between nursery areas and different levels of dependence upon the freshwater energy pathway. A diel and semi-lunar activity pattern was detected for S. senegalensis. Experimental work on gastric evacuation and feeding behaviour and its application to the wild populations allowed the estimation of food consumption by juvenile soles. Temperature, salinity and predation pressure were found to affect prey consumption. Otolith daily increments and RNA-DNA ratio analyses, showed that growth rates and condition variation reflects estuarine colonization patterns. The Tagus estuary soles were found to be in good overall condition and their growth rates were higher than at higher latitudes. The use of these methodologies for habitat quality monitoring was discussed. Estimation of spawning time through back-counting of otolith daily increments and comparison with other areas revealed a latitudinal variation in S. solea spawning in that it tends to occur earlier with decreasing latitude. Investigation on the effect of climate and hydrodynamics upon migrating sole larvae emphasized the importance of river drainage in this process. The magnitude of the mortality caused by fishing conducted within the nursery areas was considerable for S. solea and lower for S. senegalensis. Several management measures were suggested and discussed. Future studies were proposed.
Fundação para a Ciência e a Tecnologia (FCT), (SFRH/BD/12259/2003)