O ensino da biologia e geologia com recursos às tecnologias da informação e comunicação:implicações para a aprendizagem

Tese de doutoramento, Educação (Tecnologias da Informação e Comunicação na Educação), Universidade de Lisboa, Instituto de Educação, 2014

Human factors required for mycobacterium tuberculosis macrophage infection

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014

The role of alpha-synuclein phosphorylation in synucleinopathies

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014

Biological membranes : biophysical properties and aquaporin function

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

Effect of CDC25 and WEE1 on Plant Cell Cycle and Morphogenesis

The cell cycle comprise the four phases of, G1, S-phase, G2 and mitosis. Two critical transitions are G1/S and G2/M; the latter is regulated by WEE1 kinase and CDC25 phosphatases. The scope of this thesis was to investigate the regulation of the G2/M transition of the cell cycle by WEE1 and CDC25, and how these genes interface with plant growth regulators in Arabidopsis thaliana. In Arabidopsis roots, the frequency of lateral roots was found to be increased by ectopic expression of Schizosaccharomyces pombe (Sp)cdc25e and reduced by Arath;WEE1 expression. I examined the effect of Arath;WEE1 and Spcdc25 on induction of shoots and roots in Arabidopsis hypocotyls in vitro. Hypocotyl explants from two over-expressing WEE1 lines , three T-DNA insertion lines and two expressing cdc25 (Spcdc25e) lines together with wild type (WT) were cultured on two-way gradients of kinetin (Kin) and naphthyl acetic acid (NAA). Below a threshold concentration of NAA (100 ng ml-1), WEE1 repressed morphogenesis in vitro, whereas at all NAA/Kin combinations Spcdc25 promoted morphogenesis (particularly root formation) over and above that in WT. Loss of function wee1-1 cultures were very similar to WT. Quantitative data indicated a significant increase in the frequency of root formation in Spcdc25e cultures compared with WT particularly at low Kin concentrations, and WEE1oe’s repressive effect was overcome by NAA but not Kin. In conclusion, WEE1 has a repressive effect on morphogenesis in vitro that can be overcome by auxin whereas Spcd25 by-passes a cytokinin requirement for the induction of morphogenesis in vitro. The role of CDC25 and WEE1 in DNA damage responses was also analysed. Two over-expressing Arath;CDC25 lines and T-DNA mutants showed no difference to WT either in standard conditions or zeocin-supplemented treatments. However, root length was longer in Arath;CDC25oe lines treated with hydroxyurea (HU) and lateral root number was increased compared to WT. This suggests a differential response of Arath;CDC25oe in the DNA replication (HU-induced) and DNA damage (zeocin-induced) checkpoints (Chapter 5). Finally the roles of WEE1 and CDC25 in cell cycle regulation were examined using tobacco TBY-2 cell cultures expressing Arath;WEE1, Nicotiana tabacum (Nicta)WEE1 or Arath;CDC25. Whilst Nicta;WEE1 lengthened G2 of the cell cycle, Arath;WEE1 had an unusual effect of shortening G2 phase and Arath;CDC25 had no observable effect (Chapter 6).

Arabidopsis T-DNA Insertional Lines For CDC25 Are Hypersensitive to Hydroxyurea But Not to Zeocin or Salt Stress.

Background and Aims In yeasts and animals, cyclin-dependent kinases are key regulators of cell cycle progression and are negatively and positively regulated by WEE1 kinase and CDC25 phosphatase, respectively. In higher plants a full-length orthologue of CDC25 has not been isolated but a shorter gene with homology only to the C-terminal catalytic domain is present. The Arabidopis thaliana;CDC25 can act as a phosphatase in vitro. Since in arabidopsis, WEE1 plays an important role in the DNA damage/DNA replication checkpoints, the role of Arath;CDC25 in conditions that induce these checkpoints or induce abiotic stress was tested. Methods arath;cdc25 T-DNA insertion lines, Arath;CDC25 over-expressing lines and wild type were challenged with hydroxyurea (HU) and zeocin, substances that stall DNA replication and damage DNA, respectively, together with an abiotic stressor, NaCl. A molecular and phenotypic assessment was made of all genotypes Key Results There was a null phenotypic response to perturbation of Arath;CDC25 expression under control conditions. However, compared with wild type, the arath;cdc25 T-DNA insertion lines were hypersensitive to HU, whereas the Arath;CDC25 over-expressing lines were relatively insensitive. In particular, the over-expressing lines consistently outgrew the T-DNA insertion lines and wild type when challenged with HU. All genotypes were equally sensitive to zeocin and NaCl. Conclusions Arath;CDC25 plays a role in overcoming stress imposed by HU, an agent know to induce the DNA replication checkpoint in arabidopsis. However, it could not enhance tolerance to either a zeocin treatment, known to induce DNA damage, or salinity stress.

Complex Interplay Between Determinants of Pacing and Performance During 20 km Cycle Time Trials

Purpose This study examined the determinants of pacing strategy and performance during self paced maximal exercise. Methods Eight well trained cyclists completed two 20 km time trials. Power output, RPE, positive and negative affect, and iEMG activity of the active musculature were recorded every 0.5km, confidence in achieving pre-exercise goals was assessed every 5 km, and blood lactate and pH were measured post-exercise. Differences in all parameters were assessed between fastest (FAST) and slowest (SLOW) trials performed. Results Mean power output was significantly higher during the initial 90% of FAST, but not the final 10%, and blood lactate concentration was significantly higher and pH significantly lower following FAST. Mean iEMG activity was significantly higher throughout SLOW. RPE was similar throughout both trials, but participants had significantly more positive affect and less negative affect throughout FAST. Participants grew less confident in their ability to achieve their goals throughout SLOW. Conclusions The results suggest that affect may be the primary psychological regulator of pacing strategy and that higher levels of positivity and lower levels of negativity may have been associated with a more aggressive strategy during FAST. Although the exact mechanisms through which affect acts to influence performance are unclear, it may determine the degree of physiological disruption that can be tolerated, or be reflective of peripheral physiological status in relation to the still to be completed exercise task.

Gene Dosage Effect of WEE1 on Growth and Morphogenesis from Arabidopsis Hypocotyl Explants.

Background and Aims How plant cell-cycle genes interface with development is unclear. Preliminary evidence from our laboratory suggested that over-expression of the cell cycle checkpoint gene, WEE1, repressed growth and development. Here the hypothesis is tested that the level of WEE1 has a dosage effect on growth and development in Arabidospis thaliana. To do this, a comparison was made of the development of gain- and loss-of-function WEE1 arabidopsis lines both in vivo and in vitro. Methods Hypocotyl explants from an over-expressing Arath;WEE1 line (WEE1oe), two T-DNA insertion lines (wee1-1 and wee1-4) and wild type (WT) were cultured on two-way combinations of kinetin and naphthyl acetic acid. Root growth and meristematic cell size were also examined. Key Results Quantitative data indicated a repressive effect in WEE1oe and a significant increase in morphogenetic capacity in the two T-DNA insertion lines compared with WT. Compared with WT, WEE1oe seedlings exhibited a slower cell-doubling time in the root apical meristem and a shortened primary root, with fewer laterals, whereas there were no consistent differences in the insertion lines compared with WT. However, significantly fewer adventitious roots were recorded for WEE1oe and significantly more for the insertion mutant wee1-1. Compared with WT there was a significant increase in meristem cell size in WEE1oe for all three ground tissues but for wee1-1 only cortical cell size was reduced. Conclusions There is a gene dosage effect of WEE1 on morphogenesis from hypocotyls both in vitro and in vivo.

Can the Revised UK Code Direct Practice?

The Nursing and Midwifery Council, the United Kingdom regulator of nursing and midwifery has recently revised its professional code of practice. This paper begins by arguing that a professional code must be capable of sustaining close reading and of action guidance. Using four exemplar clauses it is argued that the new revised code does not met this purpose. First, I show that in setting out requirements for consent and documentation, the meaning of the relevant clause has changed significantly during the editing process so that a literal reading of the final document bears little relation to established professional practice. Second, I argue that the clause concerning the nature of professional relationships has also been altered during the editing process so that it is inconsistent with other professional groups and established accounts of the professional nurse-patient relationship. Third, I argue that the clause concerning disclosure of confidential information, which survived revision and editing with its meaning intact is nevertheless factually incorrect and inconsistent with UK law and authoritative guidance. Finally, fourth, I argue that use of the word ‘inappropriate’ is inappropriate as it amounts to meaningless circularity, discussed in relation to a clause on expressing personal beliefs. Taken together, these examples demonstrate that the Code is seriously flawed and does not fulfil its purpose. One way that simple prescriptive clauses in the Code can be usefully understood is through the provision of detailed guidance. I argue that the NMC has changed its position on its view of the value of guidance and has significantly reduced the amount of written guidance and advice is provides. The paper concludes by arguing that in order to meet its action directing function, further clarifying revision and the provision of detailed guidance is required.

Synaptic dysfunction in early encephalopathies : from genes to function

Tese de doutoramento, Medicina (Neurologia), Universidade de Lisboa, Faculdade de Medicina, 2015

Towards the therapeutic use of regulatory T cells for the treatment of human autoimmune diseases

Tese de doutoramento, Medicina (Imunologia Clínica), Universidade de Lisboa, Faculdade de Medicina, 2016

Can illusory deviant stimuli be used as attentional distractors to record vMMN in a passive three stimulus oddball paradigm?

A passive three stimulus oddball paradigm was used to investigate Visual Mismatch Negativity (vMMN) a component of the Event Related Potential (ERP) believed to represent a central pre-attentive change mechanism. Responses to a change in orientation were recorded to monochrome stimuli presented to subjects on a computer screen. One of the infrequent stimuli formed an illusory figure (Kanizsa Square) aimed to capture spatial attention in the absence of an active task. Nineteen electrodes (10-20 system) were used to record the electroencephalogram in fourteen subjects (ten females) mean age 34.5 years. ERPs to all stimuli consisted of a positive negative positive complex recorded maximally over lateral occipital areas. The negative component was greater for deviant and illusory deviant compared to standard stimuli in a time window of 170-190 ms. A P3a component over frontal/central electrodes to the illusory deviant but not to the deviant stimulus suggests the illusory figure was able to capture attention and orientate subjects to the recording. Subtraction waveforms revealed visual discrimination responses at occipital electrodes, which may represent vMMN. In a control study with 13 subjects (11 females; mean age 29.23 years), using an embedded active attention task, we confirmed the existence of an earlier (150-170 ms) and attenuated vMMN. Recordings from an intracranial case study confirmed separation of N1 and discrimination components to posterior and anterior occipital areas, respectively. We conclude that although the illusory figure captured spatial attention in its own right it did not draw sufficient attentional resources from the standard-deviant comparison as revealed when using a concurrent active task.

An exploration of pre-attentive visual discrimination using event-related potentials

The Mismatch Negativity (MMN) has been characterised as a ‘pre-attentive’ component of an Event-Related Potential (ERP) that is related to discriminatory processes. Although well established in the auditory domain, characteristics of the MMN are less well characterised in the visual domain. The five main studies presented in this thesis examine visual cortical processing using event-related potentials. Novel methodologies have been used to elicit visual detection and discrimination components in the absence of a behavioural task. Developing paradigms in which a behavioural task is not required may have important clinical applications for populations, such as young children, who cannot comply with the demands of an active task. The ‘pre-attentive’ nature of visual MMN has been investigated by modulating attention. Generators and hemispheric lateralisation of visual MMN have been investigated by using pertinent clinical groups. A three stimulus visual oddball paradigm was used to explore the elicitation of visual discrimination components to a change in the orientation of stimuli in the absence of a behavioural task. Monochrome stimuli based on pacman figures were employed that differed from each other only in terms of the orientation of their elements. One such stimulus formed an illusory figure in order to capture the participant’s attention, either in place of, or alongside, a behavioural task. The elicitation of a P3a to the illusory figure but not to the standard or deviant stimuli provided evidence that the illusory figure captured attention. A visual MMN response was recorded in a paradigm with no task demands. When a behavioural task was incorporated into the paradigm, a P3b component was elicited consistent with the allocation of attentional resources to the task. However, visual discrimination components were attenuated revealing that the illusory figure was unable to command all attentional resources from the standard deviant transition. The results are the first to suggest that the visual MMN is modulated by attention. Using the same three stimulus oddball paradigm, generators of visual MMN were investigated by recording potentials directly from the cortex of an adolescent undergoing pre-surgical evaluation for resection of a right anterior parietal lesion. To date no other study has explicitly recorded activity related to the visual MMN intracranially using an oddball paradigm in the absence of a behavioural task. Results indicated that visual N1 and visual MMN could be temporally and spatially separated, with visual MMN being recorded more anteriorly than N1. The characteristic abnormality in retinal projections in albinism afforded the opportunity to investigate each hemisphere in relative isolation and was used, for the first time, as a model to investigate lateralisation of visual MMN and illusory contour processing. Using the three stimulus oddball paradigm, no visual MMN was elicited in this group, and so no conclusions regarding the lateralisation of visual MMN could be made. Results suggested that both hemispheres were equally capable of processing an illusory figure. As a method of presenting visual test stimuli without conscious perception, a continuous visual stream paradigm was developed that used a briefly presented checkerboard stimulus combined with masking for exploring stimulus detection below and above subjective levels of perception. A correlate of very early cortical processing at a latency of 60-80 ms (CI) was elicited whether stimuli were reported as seen or unseen. Differences in visual processing were only evident at a latency of 90 ms (CII) implying that this component may represent a correlate of visual consciousness/awareness. Finally, an oddball sequence was introduced into the visual stream masking paradigm to investigate whether visual MMN responses could be recorded without conscious perception. The stimuli comprised of black and white checkerboard elements differing only in terms of their orientation to form an x or a +. Visual MMN was not recorded when participants were unable to report seeing the stimulus. Results therefore suggest that behavioural identification of the stimuli was required for the elicitation of visual MMN and that visual MMN may require some attentional resources. On the basis of these studies it is concluded that visual MMN is not entirely independent of attention. Further, the combination of clinical and non-clinical investigations provides a unique opportunity to study the characterisation and localisation of putative mechanisms related to conscious and non-conscious visual processing.

Cappadocia: where Greek prepositions come to die

The Cappadocian variety of Ulaghátsh is unique among the Greek-speaking world in having lost the inherited preposition ‘se’. The innovation is found with both locative and allative uses and has af-ected both syntactic contexts in which ‘se’ was originally found, that is, as a simple preposition (1) and as the left-occurring member of circumpositions of the type ‘se’ + NP + spatial adverb (2). (1) a. tránse ci [to meidán] en ávʝa see.PST.3SG COMP ART.DEF.SG.ACC yard.SG.ACC COP.3 game.PL.NOM ‘he saw that in the yard is some game’ (Dawkins 1916: 348) b. ta erʝó da qardáʃa évɣan [to qonáq] ART.DEF.PL.NOM two ART.DEF.PL.NOM friend.PL.NOM ascend.PST.3PL ART.DEF.SG.ACC house.SG.ACC ‘the two friends went up to the house’ (Dawkins 1916: 354) (2) émi [ta qonáca mésa], kiríʃde [to ʝasdɯ́q píso] enter.PST.3SG ART.DEF.PL.NOM house.PL.ACC inside hide.PST.3SG. ART.DEF.SG.ACC cushion.SG.ACC behind ‘he went into the houses and hid behind the cushions’ (Dawkins 1916: 348) In this paper, we set out to provide (a) a diachronic account of the loss of ‘se’ in Asia Minor Greek, and (b) a synchronic analysis of its ramifications for the encoding of the semantic and grammatical functions it had prior to its loss. The diachronic development of ‘se’ is traced by comparing the Ulaghátsh data with those obtained from Cappadocian varieties that have neither lost it nor do they show signs of losing it and, crucially, also from varieties in which ‘se’ is in the process of being lost. The comparative analysis shows that the loss first became manifest in circumpositions in which ‘se’ was preposed to the complement to which in turn a wide range of adverbs expressing topological relations were postposed (émi sa qonáca mésa > émi ta qonáca mésa). This finding is accounted for in terms of Sinha and Kuteva’s (1995) distributed spatial semantics framework, which accepts that the elements involved in the constructions under investigation—the verb (émi), ‘se’ and the spatial adverb (mésa)—all contribute to the expression of the spatial relational meaning but with differences in weighting. Of the three, ‘eis’ made the most minimal contribution, the bulk of it being distributed over the verb and the adverb. This allowed for it to be optionally dropped from circumpositions, a stage attested in Phlo-tá Cappadocian and Silliot, and to be later completely abandoned, originally in allative and subsequently in locative contexts (earlier: évɣan so qonáq > évɣan to qonáq; later: so meidán en ávʝa > to meidán en ávʝa). The earlier loss in allative contexts is also dealt with in distributed semantics terms as verbs of motion such as έβγαν are semantically more loaded than vacuous verbs like the copula and therefore the preposition could be left out in the former context more easily than in the latter. The analysis also addresses the possibility that the loss of ‘se’ may ultimately originate in substandard forms of Medieval Greek, which according to Tachibana (1994) displayed SPATIAL ADVERB + NP constructions. Applying the semantic map model (Croft 2003, Haspelmath 2003), the synchronic analysis of the varieties that retain ‘se’ reveals that—like many other allative markers crosslinguistically—it displays a pattern of multifunctionality in expressing nine different functions (among others allative, locative, recipient, addressee, experiencer), which can be mapped against four domains, viz. the spatiotemporal, the social, the mental and the logicotextual (cf. Rice & Kabata 2007). In Ulaghátsh Cappadocian, none of these functions is overtly marked as such. In cases like (1), the intended spatial relational meaning is arrived at through the combination of the syntax and the inherent semantics of the verb and the zero-marked NP as well as from the context. In environments of the type exemplified by (2), the adverb contributes further to the correct interpretation. The analysis additionally shows that, despite the loss of ‘se’, Ulaghátsh patterns with all other Cappadocian varieties in one important aspect: Goal and Location are expressed similarly (by zero in Ulaghátsh, by ‘se’ in the other varieties) whereas Source is being kept distinct (expressed by ‘apó’ in all varieties). Goal-Location polysemy is very common across the world’s languages and, most crucially, prevails over other possible polysemies in the tripartite distinction Source—Location—Goal (Lestrade 2010, Nikitina 2009). Taking into account this empirical observation, our findings suggest that the reor-anisation of spatial systems can have a local effect—in our case the loss of a member of the prepositional paradigm—but will keep the original global picture intact, thus conforming to crosslinguistically robust tendencies.  References Croft, W. 2001. Radical Construction Grammar: Syntactic Theory in Typological Perspective. Oxford: Oxford University Press. Dawkins, R. M. 1916. Modern Greek in Asia Minor: A Study of the Dialects of Sílli, Cappadocia and Phárasa with Grammar, Texts, Translations and Glossary. Cambridge: Cambridge University Press. Haspelmath, M. 2003. The geometry of grammatical meaning: semantic maps and cross-linguistic comparison. In M. Tomasello (Ed.), The New Psychology of Language, Volume 2. New York: Erlbaum, 211–243. Lestrade, S. 2010. The Space of Case. Doctoral dissertation. Radboud University Nijmegen. Nikitina, T. 2009. Subcategorization pattern and lexical meaning of motion verbs: a study of the source/goal ambiguity. Linguistics 47, 1113–1141. Rice, S. & K. Kabata. 2007. Cross-linguistic grammaticalization patterns of the allative. Linguistic Typology 11, 451–514. Sinha, C. & T. Kuteva. 1995. Distributed spatial semantics. Nordic Journal of Linguistics 18:2, 167–199. Tachibana, T. 1994. Syntactic structure of spatial expressions in the “Late Byzantine Prose Alexander Romance”. Propylaia 6, 35–51.

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.