The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism.

Smooth muscle cell (SMC) proliferation is thought to play a major role in vascular restenosis after angioplasty and is a serious complication of the procedure. Developing antisense (AS) oligonucleotides as therapeutics is attractive because of the potentially high specificity of binding to their targets, and several investigators have reported inhibition of SMC proliferation in vitro and in vivo by using AS strategies. We report here the results of our experiments on vascular SMCs using AS oligonucleotides directed toward c-myb and c-myc. We found that significant inhibition of SMC proliferation occurred with these specific AS sequences but that this inhibition was clearly not via a hybridization-dependent AS mechanism. Rather, inhibition was due to the presence of four contiguous guanosine residues in the oligonucleotide sequence. This was demonstrated in vitro in primary cultures of SMCs and in arteries ex vivo. The ex vivo model developed here provides a rapid and effective system in which to screen potential oligonucleotide drugs for restenosis. We have further explored the sequence requirements of this non-AS effect and determined that phosphorothioate oligonucleotides containing at least two sets of three or four consecutive guanosine residues inhibit SMC proliferation in vitro and ex vivo. These results suggest that previous AS data obtained using these and similar, contiguous guanosine-containing AS sequences be reevaluated and that there may be an additional class of nucleic acid compounds that have potential as antirestenosis therapeutics.

Comparison of the defensive elicitation activity of cerato-populin and cerato-platanin: a structural model

Plants can defend themselves from potential pathogenic microorganisms relying on a complex interplay of signaling pathways: activation of the MAPK cascade, transcription of defense related genes, production of reactive oxygen species, nitric oxide and synthesis of other defensive compounds such as phytoalexins. These events are triggered by the recognition of pathogen’s effectors (effector-triggered immunity) or PAMPs (PAMP-triggered immunity). The Cerato Platanin Family (CPF) members are Cys-rich proteins secreted and localized on fungal cell walls, involved in several aspects of fungal development and pathogen-host interactions. Although more than hundred genes of the CPF have been identified and analyzed, the structural and functional characterization of the expressed proteins has been restricted only to few members of the family. Interestingly, those proteins have been shown to bind chitin with diverse affinity and after foliar treatment they elicit defensive mechanisms in host and non-host plants. This property turns cerato platanins into interesting candidates, worth to be studied to develop new fungal elicitors with applications in sustainable agriculture. This study focus on cerato-platanin (CP), core member of the family and on the orthologous cerato-populin (Pop1). The latter shows an identity of 62% and an overall homology of 73% with respect to CP. Both proteins are able to induce MAPKs phosphorylation, production of reactive oxygen species and nitric oxide, overexpression of defense’s related genes, programmed cell death and synthesis of phytoalexins. CP, however, when compared to Pop1, induces a faster response and, in some cases, a stronger activity on plane leaves. Aim of the present research is to verify if the dissimilarities observed in the defense elicitation activity of these proteins can be associated to their structural and dynamic features. Taking advantage of the available CP NMR structure, Pop1’s 3D one was obtained by homology modeling. Experimental residual dipolar couplings and 1H, 15N, 13C resonance assignments were used to validate the model. Previous works on CPF members, addressed the highly conserved random coil regions (loops b1-b2 and b2-b3) as sufficient and necessary to induce necrosis in plants’ leaves: that region was investigated in both Pop1 and CP. In the two proteins the loops differ, in their primary sequence, for few mutations and an insertion with a consequent diversification of the proteins’ electrostatic surface. A set of 2D and 3D NMR experiments was performed to characterize both the spatial arrangement and the dynamic features of the loops. NOE data revealed a more extended network of interactions between the loops in Pop1 than in CP. In addition, in Pop1 we identified a salt bridge Lys25/Asp52 and a strong hydrophobic interaction between Phe26/Trp53. These structural features were expected not only to affect the loops’ spatial arrangement, but also to reduce the degree of their conformational freedom. Relaxation data and the order parameter S2 indeed highlighted reduced flexibility, in particular for loop b1-b2 of Pop1. In vitro NMR experiments, where Pop1 and CP were titrated with oligosaccharides, supported the hypothesis that the loops structural and dynamic differences may be responsible for the different chitin-binding properties of the two proteins: CP selectively binds tetramers of chitin in a shallow groove on one side of the barrel defined by loops b1-b2, b2-b3 and b4-b5, Pop1, instead, interacts in a non-specific fashion with oligosaccharides. Because the region involved in chitin-binding is also responsible for the defense elicitation activity, possibly being recognized by plant's receptors, it is reasonable to expect that those structural and dynamic modifications may also justify the different extent of defense elicitation. To test that hypothesis, the initial steps of a protocol aimed to the identify a receptor for CP, in silico, are presented.

Behavioral expression of emotions and non-invasive assessment of physiological correlates in dogs (Canis familiaris)

The aim of this thesis was to validate the use of infrared thermography (IRT) to non-invasively measure emotional reactions to different situations in pet dogs (Canis familiaris). A preliminary test, aimed to evaluate the correlation between eye-temperature and rectal temperature in dog, was performed. Then, in three different situations, negative (veterinary visit), positive (palatable food rewards), and mildly stressing followed by mildly positive (separation from and reunion with the owner), variations in heat emitted from lacrimal caruncle (referred to as eye temperature) were measured with an infrared thermographic camera. In addition, heart rate and heart rate variability parameters were collected using a non-invasive heart rate monitor designed for human use and validated on dogs. All experiments were video recorded to allow behavioral coding. During the negative situation dogs’ level of activity and stress related behaviors varied across compared to the baseline and dogs showed an increase in eye temperature despite having a significant decrease in the level of activity. The positive situation was characterized by a peak in eye temperature and mean HR and dogs engaged in behaviors indicating a positive arousal, focusing on food treats and tail wagging but there were not variations in HRV during stimulation but only an increment in SDNN immediately after the stimulus. In the separation from and reunion with the owner dogs’ eye temperature and mean HR did not vary neither in the stressful nor in the positive situations, RMSSD increased after the positive episode, SDNN dropped during the two stimulations and it increased after the stimulations. During the separation from the owner dogs were mainly directed to the door or to the experimenter while during the reunion with the owner dogs were focused mainly on the owner and on the environment, exhibiting safe base effect. A different approach was used to assess the welfare of shelter dogs. Dogs were implanted with a telemeter and after implantation dogs were housed in sequence in four different situations lasting 1 week: alone, alone with toys and a stretch cot for sleeping, with an unknown, spayed, female, and alone with a daily 2-hours interaction with an experimenter. Two different approaches were tried: partially random extracted fragments from every week, behaviors from 8 a.m. to 4 p.m. were continuous during baseline and the female situation. Results showed different reactions by dogs to the different situations and interestingly not all enrichments were enjoyed by the dogs improving their welfare. Overall results suggest that IRT may represent a useful tool to investigate emotional reactions in dogs. Nevertheless, further research is needed to establish the specificity and sensivity of IRT in this context and to assess how different dogs’ characteristics, breed, previous experience and the valence and arousal elicited by the stimulus could influence the magnitude and type of the response. The role of HRV in understanding emotional valence and the one of telemeters in understanding long-term effects on sheltered dogs’ welfare is also discussed.

Effect of magnetic activity saturation in chromospheric flux-flux relationships

We present a homogeneous study of chromospheric and coronal flux–flux relationships using a sample of 298 late-type dwarf active stars with spectral types F to M. The chromospheric lines were observed simultaneously in each star to avoid spread as a result of long-term variability. Unlike other works, we subtract the basal chromospheric contribution in all the spectral lines studied. For the first time, we quantify the departure of dMe stars from the general relations. We show that dK and dKe stars also deviate from the general trend. Studying the flux–colour diagrams, we demonstrate that the stars deviating from the general relations are those with saturated X-ray emission and we show that these stars also present saturation in the Hα line. Using several age spectral indicators, we show that these are younger stars than those following the general relationships. The non-universality of flux–flux relationships found in this work should be taken into account when converting between fluxes in different chromospheric activity indicators.

Carbon and nitrogen limitation increase chitosan antifungal activity in Neurospora crassa and fungal human pathogens

Chitosan permeabilizes plasma membrane and kills sensitive filamentous fungi and yeast. Membrane fluidity and cell energy determine chitosan sensitivity in fungi. A five-fold reduction of both glucose (main carbon (C) source) and nitrogen (N) increased 2-fold Neurospora crassa sensitivity to chitosan. We linked this increase with production of intracellular reactive oxygen species (ROS) and plasma membrane permeabilization. Releasing N. crassa from nutrient limitation reduced chitosan antifungal activity in spite of high ROS intracellular levels. With lactate instead of glucose, C and N limitation increased N. crassa sensitivity to chitosan further (4-fold) than what glucose did. Nutrient limitation also increased sensitivity of filamentous fungi and yeast human pathogens to chitosan. For Fusarium proliferatum, lowering 100-fold C and N content in the growth medium, increased 16-fold chitosan sensitivity. Similar results were found for Candida spp. (including fluconazole resistant strains) and Cryptococcus spp. Severe C and N limitation increased chitosan antifungal activity for all pathogens tested. Chitosan at 100 μg ml-1 was lethal for most fungal human pathogens tested but non-toxic to HEK293 and COS7 mammalian cell lines. Besides, chitosan increased 90% survival of Galleria mellonella larvae infected with C. albicans. These results are of paramount for developing chitosan as antifungal.

Childhood Physical Activity Involvement in Active and Inactive Female Adults

Current trends show progressive declines in levels of physical activity from childhood through adolescence and adulthood, most notably for females. The current study examined organized activity involvement in active and inactive females (age 18) using retrospective data. Results indicated that active females participated in significantly more physical activities than inactive females from age 6 to age 18. No significant differences were found between groups for non-physical activities. In addition, parents of active and inactive females were the most influential factor in initiating physical activity. However, parents of active females initiated more physical activity involvement than did parents of inactive females. Results also indicate that certain periods in childhood and adolescence appear to be critical for developing long-term physical activity habits.

Role of Dopamine D2 Receptors in the Consolidation of Amphetamine-Cue Memory in Conditioned Activity in Rats

The neurotransmitter dopamine (DA) plays an essential role in reward-related incentive learning, whereby neutral stimuli gain the ability to elicit approach and other responses. In an incentive learning paradigm called conditioned activity, animals receive a stimulant drug in a specific environment over the course of several days. When then placed in that environment drug-free, they generally display a conditioned hyperactive response. Modulating DA transmission at different time points during the paradigm has been shown to disrupt or enhance conditioning effects. For instance, blocking DA D2 receptors before sessions generally impedes the acquisition of conditioned activity. To date, no studies have examined the role of D2 receptors in the consolidation phase of conditioned activity; this phase occurs immediately after acquisition and involves the stabilization of memories for long-term storage. To investigate this possible role, I trained Wistar rats (N = 108) in the conditioned activity paradigm produced by amphetamine (2.0 mg/kg, intraperitoneally) to examine the effects of the D2 antagonist haloperidol (doses 0.10, 0.25, 0.50, 0.75, 1.0, & 2.0 mg/kg, intraperitoneally) administered 5 min after conditioning sessions. Two positive control groups received haloperidol 1 h before conditioning sessions (doses 1.0 mg/kg and 2.0 mg/kg). The results revealed that post-session haloperidol at all doses tested did not disrupt the consolidation of conditioned activity, while pre-session haloperidol at 2.0 mg/kg prevented acquisition, with the 1.0 mg/kg group trending toward a block. Additionally, post-session haloperidol did not diminish activity during conditioning days, unlike pre-session haloperidol. One possible reason for these findings is that the consolidation phase may have begun earlier than when haloperidol was administered, since the conditioned activity paradigm uses longer learning sessions than those generally used in consolidation studies. Future studies may test if conditioned activity can be achieved with shorter sessions; if so, haloperidol would then be re-tested at an earlier time point. D2 receptor second messenger systems may also be investigated in consolidation. Since drug-related incentive stimuli can evoke cravings in those with drug addiction, a better understanding of the mechanisms of incentive learning may lead to the development of solutions for these individuals.

The concept of 'economic activity' in the EU treaty: from ideological dead-ends to workable judicial concepts. Research Paper in Law 06/2011

[From the Introduction]. The economic rules, or put more ambitiously, the economic constitution of the Treaty,1 only apply to economic activities. This general principle remains valid, even if some authors strive to demonstrate that certain Treaty rules also apply in the absence of an economic activity,2 and despite the fact that non-economic (horizontal) Treaty provisions (e.g. principle of nondiscrimination, rules on citizenship) are also applicable in the absence of any economic activity.3 Indeed, the exercise of some economic activity transcends the concepts of ‘goods’ (having positive or negative market value),4 workers (even if admitted in an extensive manner),5 and services (offered for remuneration).6 It is also economic activity or ‘the activity of offering goods and services into the market’7 that characterises an ‘undertaking’ thus making the competition rules applicable. Further, it is for regulating economic activity that Article 115 TFEU, Article 106(3) TFEU and most other legal bases in the TFEU provide harmonisation powers in favour of the EU. Last but not least, Article 14 TFEU on the distinction between services of general economic interest (SGEIs) and non-economic services of general interest (NESGIs), as well as Protocol n. 26 on Services of General Interest (SGIs) confirm the constitutional significance of the distinction between economic and non-economic: a means of dividing competences between the EU and the member states. The distinction between economic and non-economic activities is fraught with legal and technical intricacies – the latter being generated by dynamic technological advances and regulatory experimentation. More importantly, however, the distinction is overcharged with political and ideological significations and misunderstandings and, even, terminological confusions.8

The antioxidant activity of novel chromones and xanthones: a structure-activity relationship

Chromones and xanthones are oxygen-containing heterocyclic compounds with bioactive properties widely reported in the literature, specially concerning to their antioxidant properties. The search for new natural and synthetic chromone and xanthone derivatives order to evaluate and discover new structural features rendering optimized biological effects has been a challenge. Thus, the aim of this work was to evaluate the scavenging activity of reactive oxygen (ROS) and nitrogen (RNS) species of new synthetic hydroxylated chromones and xanthones (Fig. 1) using in vitro non-cellular systems. These compounds exhibited scavenger effects dependent on the concentration, with IC50 values found at the micromolar range. The overall scavenging activity of chromones was better than xanthones, specially the one of chromone 3A. In conclusion, the novel tested chromone and xanthone scaffolds proved to be promising pharmacophores with potential therapeutic applications as antioxidant agents.

Mycophenolatemofetil in non-renal systemiclupuserythematosusmanifestations

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Patterns and Determinants of Off-Farm Migration: Transfer frictions and persistency of relative income gaps. Factor Markets Working Papers No. 36, February 2013

The inter-sectoral migration of agricultural labour is a complex but fundamental process of economic development largely affected by the growth of agricultural productivity and the evolution of the agricultural relative income gap. Theory and some recent anecdotal evidence suggest that as an effect of large fixed and sunk costs of out-farm migration, the productivity gap between the agricultural and non-agricultural sectors should behave non-monotonically or following a U-shaped evolution during economic development. Whether or not this relationship holds true across a sample of 38 developing and developed countries and across more than 200 EU regions was empirically tested. Results strongly confirm this relationship, which also emphasises the role played by national agricultural policy.

The Role of Pluriactivity in Farm Exit and Labour Supply Decisions. Factor Markets Working Document No. 67, August 2013

EDITED VERSION TO BE PUBLISHED SOON Pluriactivity has been a topic of research in agriculture for the best part of a century. It is a term which has both broad and narrow definitions and hence is subject to multiple interpretations. This paper considers two forms of pluriactivity: within the farm gate pluriactivity, also commonly referred to as farm diversification, and beyond the farm-gate pluriactivity, also known as multiple job holding. Previous studies of pluriactivity have shown that it can inhibit the natural process of structural change in the farm sector, by allowing small and unprofitable farms to survive with the support of income from outside the sector. In this paper, two empirical models of pluriactivity are estimated using farm level data for Ireland. The first examines the impact of on-farm diversification on off-farm labour supply, while the second investigates the relationship between off-farm labour supply and farm exit which is specified in the context of retirement and non-succession. The result of the first model suggests that farms that engage in within the farm gate pluriactivity are less likely to engage in beyond the farm gate pluriactivity, in other words more diversified farmers are less likely to work off farm. The second model confirms previous findings in the literature that part-time farmers have a reduced probability of having a farm successor. While the model results are specific to the Irish case, they do provide some value insights into the impacts of pluriactivity on structural change in farming.

Building International CSR on Solid Foundations: Location and Network Aspects of Nonmarket Environments in Europe and Non-Europe Markets. ACES Working Papers, 2011

Multinational companies' (MNCs) corporate social responsibility (CSR) programs frequently comprise a portfolio of disconnected country-level programs or, alternatively, consist of blanket corporate policies that apply in the same way across the geographies where the company operates. Yet, the international nonmarket environment in which CSR programs operate is neither a completely fragmented nor a perfectly homogeneous one. Building on the concept of stakeholder-issue-networks, we develop a model that explicitly takes into consideration the role of geography in the characterization of a firm's nonmarket environment. This allows us to develop a taxonomy of nonmarket environments on the basis of their geographic spread and their degree of cross-border connectedness. We then explore the strategic and organizational implications that different ideal types of (cross-border) nonmarket environments have for the development of international CSR policies.

Pro-American non-alignment. Sweden and Finland develop closer military co-operation with the United States. OSW COMMENTARY NUMBER 205/31.03.2016

Russia’s increasingly aggressive policy and its enhanced military activity in the Nordic-Baltic region has led to revaluations in Sweden’s and Finland’s security and defence policies and a rethinking of the formats of their military co-operation. While remaining outside NATO, the two states have been developing closer bilateral defence co-operation and working more closely with the United States, while at the same time developing co-operation with NATO. Sweden and Finland perceive the United States as the guarantor of regional and European security. From their point of view, the United States is currently the country that has both the necessary military capabilities and the political will to react in the event of a conflict between Russia and NATO in the Nordic-Baltic region, in which both countries would inevitably become involved despite their non-aligned status. For Sweden and Finland, intensified co-operation with the United States offers an alternative to NATO membership, which is currently out of the question for domestic political reasons. Meanwhile, the US has also become increasingly aware of the strategic importance of the two states, which, for the purposes of contingency planning, are in fact an extension of NATO’s north-eastern flank.

Cdk Activity Couples Epigenetic Centromere Inheritance to Cell Cycle Progression

Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We further show that the key CENP-A assembly factor Mis18BP1(HsKNL2) is phosphorylated in a cell cycle-dependent manner that controls its centromere localization during mitotic exit. These results strongly support a model in which the CENP-A assembly machinery is poised for activation throughout the cell cycle but kept in an inactive noncentromeric state by Cdk activity during S, G2, and M phases. Alleviation of this inhibition in G1 phase ensures tight coupling between DNA replication, cell division, and subsequent centromere maturation.