Identification and validation of microsatellite markers in strawberry tree (Arbutusunedo L.)

Strawberry tree (Arbutus unedo L.), an evergreen shrub/small tree of the family Ericaceae, is a main constituent of the Mediterranean basin flora; although it is also found in southwestern Prance, Macaronesia, and Ireland. The small fruits are edible but mostly used for preparation of preserves and jams, and for liquors such as the Portuguese traditional "aguardente de medronho". Traditionally cultivated by small farmers, often in consociation with Quercus sp., strawberry tree is presently emerging as a new important fruit crop cultivated in large orchards by modern export-oriented enterprises. This change of paradigm requires a growing role of plant breeding, upstream of the production process. Genomic tools for this species are mostly limited to the chloroplast genome sequence and to genomic data described in this work. In order to identify strawberry tree microsatellite (SSR) loci we performed partial genome next-generation sequencing using the Ion Torrent technology. The sequenced similar to 24.6M nucleotides resulted in the identification of 1185 microsatellite markers mostly constituted by dinucleotide motifs. The relative amount of microsatellite dinucleotide motifs (AG/CT - 71.7%, AC/GT - 20.5%, AT/AT - 2.9%, and CG/CG - 0.3%) is similar to the one observed in other Ericaceae species. Among a tested sample of 40 SSR primer pairs, 20 amplified well-defined PCR products, 12 (30%) were validated as polymorphic. Used in our collaborative project for molecular identification of selected and improved clones, the identified SSR loci constitute a strong tool for a large panoply of applied and fundamental studies of this emerging fruit crop.Pluriannual Funding Program of the Portuguese National Foundation for Science and Technologyinfo:eu-repo/semantics/publishedVersio

Role of the neurotrophic factor receptor RET in haematopoiesis

Tese de doutoramento, Ciências Biomédicas (Imunologia), Universidade de Lisboa, Faculdade de Medicina, 2013Haematopoiesis is a developmental process that ensures the generation of all blood cell lineages throughout life. As a consequence, this is a highly complex and dynamic developmental cascade subject to tight regulatory mechanisms. Thus, the study of novel molecular signals is critical to further understand how haematopoiesis operates to ensure the balance between cell lineage commitment, cell homeostasis and efficient haematopoietic responses to insults and disturbances. The tyrosine kinase RET is the receptor for the GDNF (glial cell line-derived neurotrophic factor) neurotrophic factor family (GDNF family ligands – GFLs). Productive RET signalling controls the development and maintenance of the enteric nervous system, kidneys and spermatogenesis. Interestingly, Ret expression was detected in haematopoietic cells and lymphoid organs and RET signalling was shown to regulate enteric lymphoid organogenesis. However the role of RET in haematopoiesis remains completely unexplored. Haematopoietic stem cells (HSCs) are at the onset of the developmental cascade that generate all blood cells, thus we initially investigated the role of RET in HSC function and how modulation of RET signalling can be used to control HSC responses. Finally, we investigated the role of RET in late stages of haematopoietic cell precursor differentiation into the T cell lineage. We found that the tyrosine kinase RET is critical to HSC survival and function. HSCs express RET signalling molecules and HSCs microenvironment provides RET ligands. Moreover Ret ablation leads to reduced HSC numbers and recruitment of quiescent cells into proliferation. Although RET null progenitors have normal differentiation potential, they exhibit impaired in vivo stress response and reconstitution potential. Remarkably RET downstream signalling results in p38/MAP kinase phosphorylation and CREB transcription factor activation, providing HSCs with critical surviving cues. In agreement, recue of Ret null progenitors was efficiently achieved in vivo by forcing the expression of RET downstream targets, Bcl2 or Bcl2l1. Thus, RET activation improves HSC survival and in vivo transplantation efficiency, unveiling exciting new possibilities in transplantation and HSC ex vivo expansion. In addition, our work demonstrates that HSC use neurotrophic factors to regulate and maintain their fitness. RET signalling molecules are also expressed in thymocytes, more specifically, we found their expression in CD4/CD8 double negative thymocytes (DN). Nevertheless, ablation of Ret or it co-receptors Gfra1 and Gfra2 had a minor impact in foetal thymopoiesis. In agreement, Ret conditional knockout mice had similar thymocyte development and fitness when compared to their WT counterparts. Thus, while RET signalling is critical to HSC function, it is dispensable for T cell development in vivo. Altogether, our work show that molecular mechanisms usually assigned to specific tissues, can be more widely used by unrelated cell types, such as haematopoietic stem cells. Our findings also illustrate how a same signalling pathway can be regulated to originate different cell responses. Unlike several neuronal populations that use GFLs depending on the specific expressed co-receptor, HSCs express multiple RET coreceptors and respond to GFLs in a redundant fashion. There is increasing evidence that nervous signals can control haematopoiesis, namely by regulating osteoblast and mesenchymal stem cell function in HSC niches. Herein, we show that neurons and HSC employ common regulatory mechanisms. Thus, our work paves the way to further studies employing neurotrophic factors in HSC expansion and transplantation protocols.O sistema hematopoiético, que inclui as células do sistema imunitário, é altamente complexo e dinâmico, e como tal, está sujeito a uma regulação apertada. Assim, o estudo dos mecanismos moleculares responsáveis pelo desenvolvimento de células hematopoiéticas é essencial para compreender a forma como este sistema funciona de modo a manter o equilíbrio entre o número de células necessárias em homeostasia e uma rápida resposta em situações de desequilíbrio. A tirosina cinase RET é o receptor para os factores neurotróficos da família do GDNF (glial cell line-derived neurotrophic factor) (GDNF family ligands – GFLs) e tem uma função crucial no desenvolvimento e manutenção do sistema nervoso, no desenvolvimento embrionário do rim e na espermatogénese. Curiosamente, a expressão de Ret já foi detectada em várias populações de células hematopoiéticas e em órgãos linfoides primários como o fígado fetal, a medula óssea e o timo. Para além disso, foi também demonstrado um papel crucial da sinalização por RET em células hematopoiéticas envolvidas na organogénese das placas de Peyer no intestino durante a vida embrionária. No entanto, apesar de a expressão de RET ter sido identificada em várias populações celulares, uma possível função de RET no desenvolvimento ou função de células hematopoiéticas não é ainda conhecida. As células estaminais hematopoiéticas, pelas suas capacidades de autorrenovação e proliferação, são a base do programa de desenvolvimento e diferenciação que origina todas as células do sangue. Por este motivo, é crucial compreender os complexos mecanismos que regulam as células estaminais hematopoiéticas. Assim, no laboratório estamos particularmente interessados no papel de RET na função das células estaminais hematopoiéticas e no modo como a sinalização por RET pode ser usada de forma a modular a sua resposta em situações de desequilíbrio como a transplantação. Uma vez que foi proposto um efeito do ligando de RET GDNF na sobrevivência de timócitos in vitro, estamos também interessados em compreender a função de RET no desenvolvimento de células T in vivo. O timo é responsável pela produção de todas as populações de células T, que são essenciais para respostas imunitárias eficientes. Como tal é indispensável perceber de que modo o microambiente tímico fornece uma complexa rede de sinais que levam ao desenvolvimento de células T a partir de precursores da medula óssea. No laboratório descobrimos que a tirosina cinase RET tem uma função crítica na sobrevivência e função das células estaminais hematopoiéticas. Estas expressam a maquinaria de sinalização por RET, que inclui o receptor RET e os seus co-receptores, enquanto o microambiente onde as células estaminais hematopoéticas se encontram providencia os ligandos de RET necessários. Para além disso, a ablação de Ret leva à redução do número de células estaminais hematopoiéticas e ao recrutamento de células quiescentes para o estado proliferativo. Apesar de os progenitores hematopoiéticos deficientes em RET terem um potencial de diferenciação normal, apresentam uma fraca resposta ao stresse in vivo e um potencial de reconstituição reduzido. Importante, a sinalização de RET fornece factores de sobrevivência às células estaminais hematopoiéticas, por a jusante resultar na fosforilação da cinase p38/MAPK e na activação do factor de transcrição CREB. Em concordância, a sobreexpressão dos genes alvo a jusante de RET, Bcl2 ou Bcl2l1, resgata in vivo a função hematopoiética de progenitores deficientes em Ret, aumentado o seu potencial te transplantação. Na verdade, a activação de RET aumenta a sobrevivência de células estaminais hematopoiéticas e a sua eficiência de transplantação in vivo, revelando novas possibilidades de intervenção em terapias de transplantação e expansão ex vivo de células estaminais hematopoiéticas. Assim, o nosso trabalho mostra que factores neurotróficos presentes no nicho das células estaminais hematopoiéticas regulam a sua função através do receptor RET. Embora tenhamos confirmado que as moléculas envolvidas na sinalização de RET são expressas no timo, especialmente na população de timócitos negativa para os coreceptores CD4 e CD8 (DN), a remoção de Ret ou dos seus co-receptores Gfra1 ou Gfra2 não afecta a timopoiese fetal. Concordantemente, animais adultos com eliminação condicional de Ret em timócitos revelam que a capacidade de desenvolvimento de timócitos deficientes em Ret é semelhante à dos controlos selvagens. Do mesmo modo mutações que conferem um ganho de função no receptor RET não influenciam o desenvolvimento tímico. Assim, apesar de a sinalização por RET poder fornecer sinais de sobrevivência a timócitos, esta é dispensável para o desenvolvimento de células T in vivo, mesmo em condições de competição entre progenitores deficientes ou competentes em Ret. No seu conjunto, o nosso trabalho demonstra que mecanismos moleculares geralmente atribuídos a tecidos específicos, podem ser mais amplamente utilizado por tipos de células não relacionadas, tais como células estaminais hematopoiéticas. Os nossos resultados também ilustram como uma mesma via de sinalização pode ser 3 regulada de forma a originar diferentes respostas celulares. Contrariamente a diversas populações de neurónios, que usam GFLs específicos dependendo do coreceptor que expressam, as HSCs expressam múltiplos coreceptores de RET respondem aos GFLs de forma redundante. Existem cada vez mais evidências de que os sinais provenientes do sistema nervoso podem controlar hematopoiese, nomeadamente através da regulação da função dos osteoblastos e células estaminais mesenquimais presentes nos nichos das HSCs. Surpreendentemente, nós mostramos que os neurônios e as HSCs utilizam mecanismos comuns de regulação.. Deste modo, o nosso trabalho abre caminho a novos estudos na utilização factores neurotróficos em protocolos de expansão e transplantação de HSCs

Bound-states and polarized charged zero modes in three-dimensional topological insulators induced by a magnetic vortex

By coating a three-dimensional topological insulator (TI) with a ferromagnetic film supporting an in-plane magnetic vortex, one breaks the time-reversal symmetry (TRS) without generating a mass gap. It rather yields electronic states bound to the vortex center which have different probabilities associated with each spin mode. In addition, its associate current (around the vortex center) is partially polarized with an energy gap separating the most excited bound state from the scattered ones. Charged zero-modes also appear as fully polarized modes localized near the vortex center. From the magnetic point of view, the observation of such a special current in a TI-magnet sandwich comes about as an alternative technique for detecting magnetic vortices in magnetic thin films.Comment: 8 pages, 3 figures, new version with more discussions and results accepted for publication in The European Physical Journal

Cyclical Effects of Bank Capital Buffers with Imperfect Credit Markets: international evidence

This paper analyzes the cyclical effects of bank capital buffers using an international sample of 2,361 banks from 92 countries over the 1990-2007 period. We find that capital buffers reduce the bank credit supply but – through what could be “monitoring or signaling effects” – have also an expansionary effect on economic activity by reducing lending and deposit rate spreads. This influence on lending and deposit rate spreads is more pronunced in developing countries and during downturns. The results suggest that capital buffers have a counter-cyclical effect in these countries. Our data do not suggest differences in the cyclical effects of capital buffers between Basel I and Basel II.

Proton Profile Function at 52.8 GeV

We present the results of a novel model-independent fit to elastic proton-proton differential cross section data at $\sqrt s$ = 52.8 GeV. Taking into account the error propagation from the fit parameters, we determine the scattering amplitude in the impact parameter space (the proton profile function) and its statistical uncertainty region. We show that both the real and imaginary parts of the profile are consistent with two dynamical contributions, one from a central dense region, up to roughly 1 fm and another from a peripheral evanescent region from 1 to 3 fm.Comment: Published in Int. J. Mod. Phys.

Evaluation of atherosclerotic plaques with nano-probes for intravascular cardiological imaging diagnosis

Cardiovascular diseases represent the leading cause of mortality and disability worldwide. Besides, it is estimated that these numbers will increase significantly in the future. More specifically, atherosclerosis is present in most of the main cardiovascular diseases, making its study urgent and important to develop new diagnostic tools. The most relevant limitation in the current investigation of the evaluation of atherosclerosis is the impossibility to distinguish stable and prone to rupture (unstable) plaques in coronary arteries. Associated with the instability phenomena, in plaques prone to rupture, are increased thickness of tissue layers (already detectable but not provide an unequivocal diagnosis) and inflammatory processes (not yet detectable but only present when the lesion evolves higher risk to rupture). Aiming to detect the precise location of inflammatory processes, two types of contrast agents (nano probes) were synthetized, gold nanoparticles and microbubbles. Also, to replicate the behavior of human arteries, three-dimensional tissue simulating structures (phantoms) were fabricated and optimized. To evaluate the performance of the contrast agents, both in the phantoms and post-mortem human arteries, optical coherence tomography (OCT) images were acquired in a clinical environment, and other techniques were performed (confocal microscopy, scanning electron microscopy, atomic force microscopy) to characterize the samples. Microbubbles revealed to be a better contrast agent than gold nanoparticles having a clearly noticeable enhancement of the OCT signal. After the acquisition of several OCT images on both types of samples (arteries and phantoms) an automatic imaging processing software was developed to detect the presence of the contrast agents and its posterior location. The software uses MATLAB as a programming language and with a user-friendly interface the user can access numerous parameters of the analyzed image and even edit them manually. In the end of the automatic processing, the user has the information of the number of regions of interest as well as their visual location

Computational modeling of prefrontal cortex circuits

Dissertation presented to obtain the Ph.D degree in BiologyThe most outstanding feature of the human brain is its ability to perform highly complex cognitive tasks and one key region of the brain involved in these elaborated tasks is the prefrontal cortex. However, little is known about the basic neuronal processes that sustain these capacities. This dissertation describes the computational study of the biophysical properties of neurons in the prefrontal cortex that underlie complex cognitive processes with special emphasis in working memory, the ability to keep information online in the brain for a short period of time while processing incoming external stimuli.(...