Molecular regulation of secondary gorwth: searching for SHORT-ROOT function in cork formation

The analysis of molecular regulators involved in controlling the maintenance and function of plant meristems has been the subject of many studies. Some master regulators of these processes have been identified in Arabidopsis benefiting from the array of tools available for genetic and molecular analysis in this model plant. However, aspects such as secondary growth that are more extensively observed in woody plants, have been less studied. Secondary growth is responsible for the enlargement of the plant stems and roots and results from the activity of the lateral (secondary) meristems, vascular cambium and cork cambium (phellogen), which produce two important renewable natural resources, wood and cork, respectively.(...)

Size-Control Regulation during Regeneration

Functional regeneration of organs upon injury is a key process for animals survival. Contrary to humans, some vertebrates are remarkably competent in regenerating after acute organ or appendage lesions. This advantageous skill allows overcoming limitations in repair even in adult stages, when tissues are fully developed, via a process of epimorphic regeneration. One such organism is the zebrafish, which can regenerate several organs, namely its heart, retina, spinal cord and fins. (...)

Characterization and regulation of a bacterial sugar phosphatase of the HAD superfamily, AraL, from Bacillus subtilis.

AraL from Bacillus subtilis is a member of the ubiquitous haloalkanoate dehalogenase, HAD, superfamily. The araL gene has been cloned, over-expressed in Escherichia coli and its product purified to homogeneity. The enzyme displays phosphatase activity, which is optimal at neutral pH (7.0) and 65 °C. Substrate screening and kinetic analysis showed AraL to have low specificity and catalytic activity towards several sugar phosphates, which are metabolic intermediates of the glycolytic and pentose phosphate pathways. Based on substrate specificity and gene context within the arabinose metabolic operon, a putative physiological role of AraL in detoxification of accidental accumulation of phosphorylated metabolites has been proposed. The ability of AraL to catabolise several related secondary metabolites requires regulation at the genetic level. Here, by site- directed mutagenesis, we show that AraL production is regulated by a structure in the translation initiation region of the mRNA, which most probably blocks access to the ribosome-binding site, preventing protein synthesis. Members of HAD subfamily IIA and IIB are characterised by a broad-range and overlapping specificity that anticipated the need for regulation at the genetic level. In this study we provide evidence for the existence of a genetic regulatory mechanism controlling AraL production.

Technology transition towards electric mobility - technology assessment as a tool for policy design

Paper presented at the Colloquium Gerpisa 2013, Paris (http://gerpisa.org/node/2085), Session n°: 19 New kinds of mobility: old and new business models

Trans-Synaptic Signaling by Activity-Dependent Cleavage of Neuroligin-1

Throughout the brain, patterns of activity in postsynaptic neurons influence the properties of synaptic inputs. Such feedback regulation is central to neural network stability that underlies proper information processing and feature representation in the central nervous system. At the cellular level, tight coupling of presynaptic and postsynaptic function is fundamental to neural computation and synaptic plasticity. The cohort of protein complexes at the pre and postsynaptic membrane allows for tight synapse-specific segregation and integration of diverse molecular and electrical signals.(...)

Exploring ionic liquids′ unique stimuli to elucidate uncharacterised cellular and molecular mechanisms in filamentous fungi

The emergence of new fungal pathogens, either of plants or animals, and the increasing number of reported cases of resistant human pathogenic strains to the available antifungal drugs reinforces the need for better understanding the biology of filamentous fungi. Conventional drugs target components of the fungal membrane or cell wall, therefore identifying novel intracellular targets, yet unique to fungi, is a global priority.(...)

Cellular reprogramming strategies for degenerative disorders involving the retinal pigment epithelium

RESUMO: A reprogramação celular permite que uma célula somática seja reprogramada para outra célula diferente através da expressão forçada de factores de transcrição (FTs) específicos de determinada linhagem celular, e constitui uma área de investigação emergente nos últimos anos. As células somáticas podem ser experimentalmente manipuladas de modo a obter células estaminais pluripotentes induzidas (CEPi), ou convertidas directamente noutro tipo de célula somática. Estas descobertas inovadoras oferecem oportunidades promissoras para o desenvolvimento de novas terapias de substituição celular e modelos de doença, funcionando também como ferramentas valiosas para o estudo dos mecanismos moleculares que estabelecem a identidade celular e regulam os processos de desenvolvimento. Existem várias doenças degenerativas hereditárias e adquiridas da retina que causam deficiência visual devido a uma disfunção no tecido de suporte da retina, o epitélio pigmentar da retina (EPR). Uma destas doenças é a Coroideremia (CHM), uma doença hereditária monogénica ligada ao cromossoma X causada por mutações que implicam a perda de função duma proteína com funções importantes na regulação do tráfico intracelular. A CHM é caracterizada pela degenerescência progressiva do EPR, assim como dos foto-receptores e da coróide. Resultados experimentais sugerem que o EPR desempenha um papel importante na patogénese da CHM, o que parece indicar uma possível vantagem terapêutica na substituição do EPR nos doentes com CHM. Por outro lado, existe uma lacuna em termos de modelos in vitro de EPR para estudar a CHM, o que pode explicar o ainda desconhecimento dos mecanismos moleculares que explicam a patogénese desta doença. Assim, este trabalho focou-se principalmente na exploração das potencialidades das técnicas de reprogramação celular no contexto das doenças de degenerescência da retina, em particular no caso da CHM. Células de murganho de estirpe selvagem, bem como células derivadas de um ratinho modelo de knockout condicional de Chm, foram convertidos com sucesso em CEPi recorrendo a um sistema lentiviral induzido que permite a expressão forçada dos 4 factores clássicos de reprogramação, a saber Oct4, Sox2, Klf4 e c-Myc. Estas células mostraram ter equivalência morfológica, molecular e funcional a células estaminais embrionárias (CES). As CEPi obtidas foram seguidamente submetidas a protocolos de diferenciação com o objectivo final de obter células do EPR. Os resultados promissores obtidos revelam a possibilidade de gerar um valioso modelo de EPR-CHM para estudos in vitro. Em alternativa, a conversão directa de linhagens partindo de fibroblastos para obter células do EPR foi também abordada. Uma vasta gama de ferramentas moleculares foi gerada de modo a implementar uma estratégia mediada por FTs-chave, seleccionados devido ao seu papel fundamental no desenvolvimento embrionário e especificação do EPR. Conjuntos de 10 ou menos FTs foram usados para transduzir fibroblastos, que adquiriram morfologia pigmentada e expressão de alguns marcadores específicos do EPR. Adicionalmente, observou-se a activação de regiões promotoras de genes específicos de EPR, indicando que a identidade transcricional das células foi alterada no sentido pretendido. Em conclusão, avanços significativos foram atingidos no sentido da implementação de tecnologias de reprogramação celular já estabelecidas, bem como na concepção de novas estratégias inovadoras. Metodologias de reprogramação, quer para pluripotência, quer via conversão directa, foram aplicadas com o objectivo final de gerar células do EPR. O trabalho aqui descrito abre novos caminhos para o estabelecimento de terapias de substituição celular e, de uma maneira mais directa, levanta a possibilidade de modelar doenças degenerativas da retina com disfunção do EPR numa placa de petri, em particular no caso da CHM.---------------ABSTRACT: Cellular reprogramming is an emerging research field in which a somatic cell is reprogrammed into a different cell type by forcing the expression of lineage-specific transcription factors (TFs). Cellular identities can be manipulated using experimental techniques with the attainment of pluripotency properties and the generation of induced Pluripotent Stem (iPS) cells, or the direct conversion of one somatic cell into another somatic cell type. These pioneering discoveries offer new unprecedented opportunities for the establishment of novel cell-based therapies and disease models, as well as serving as valuable tools for the study of molecular mechanisms governing cell fate establishment and developmental processes. Several retinal degenerative disorders, inherited and acquired, lead to visual impairment due to an underlying dysfunction of the support cells of the retina, the retinal pigment epithelium (RPE). Choroideremia (CHM), an X-linked monogenic disease caused by a loss of function mutation in a key regulator of intracellular trafficking, is characterized by a progressive degeneration of the RPE and other components of the retina, such as the photoreceptors and the choroid. Evidence suggest that RPE plays an important role in CHM pathogenesis, thus implying that regenerative approaches aiming at rescuing RPE function may be of great benefit for CHM patients. Additionally, lack of appropriate in vitro models has contributed to the still poorly-characterized molecular events in the base of CHM degenerative process. Therefore, the main focus of this work was to explore the potential applications of cellular reprogramming technology in the context of RPE-related retinal degenerations. The generation of mouse iPS cells was established and optimized using an inducible lentiviral system to force the expression of the classic set of TFs, namely Oct4, Sox2, Klf4 and c-Myc. Wild-type cells, as well as cells derived from a conditional knockout (KO) mouse model of Chm, were successfully converted into a pluripotent state, that displayed morphology, molecular and functional equivalence to Embryonic Stem (ES) cells. Generated iPS cells were then subjected to differentiation protocols towards the attainment of a RPE cell fate, with promising results highlighting the possibility of generating a valuable Chm-RPE in vitro model. In alternative, direct lineage conversion of fibroblasts into RPE-like cells was also tackled. A TF-mediated approach was implemented after the generation of a panoply of molecular tools needed for such studies. After transduction with pools of 10 or less TFs, selected for their key role on RPE developmental process and specification, fibroblasts acquired a pigmented morphology and expression of some RPE-specific markers. Additionally, promoter regions of RPE-specific genes were activated indicating that the transcriptional identity of the cells was being altered into the pursued cell fate. In conclusion, highly significant progress was made towards the implementation of already established cellular reprogramming technologies, as well as the designing of new innovative ones. Reprogramming into pluripotency and lineage conversion methodologies were applied to ultimately generate RPE cells. These studies open new avenues for the establishment of cell replacement therapies and, more straightforwardly,raise the possibility of modelling retinal degenerations with underlying RPE defects in apetri dish, particularly CHM.

Monitorização de consumos energéticos em edifícios: um estudo de caso na Schneider Electric

Na sequência do panorama económico atual, existe cada vez mais uma tendência de redução de custos e de, em simultâneo, obter a eficiência energética desejada, mais concretamente no setor dos edifícios, uma vez que este tem um peso bastante considerável nos consumos totais em Portugal e na Europa. Existem variadas formas de atuar neste sentido nomeadamente ao nível dos equipamentos e sistemas instalados, bem como, nos hábitos e comportamentos dos ocupantes. Com esse intuito, muitas empresas têm desenvolvido mecanismos ou formas de minimizar os gastos, nomeadamente em termos de consumos elétricos. Dentro dos possíveis caminhos para se alcançar esse objetivo, surgem os sistemas de monitorização e os sistemas de gestão técnica centralizada. O objetivo de reduzir a discrepância entre a fatura que se paga pela eletricidade e o que realmente consiste numa necessidade de consumo é atualmente uma preocupação real. Desta forma, o desenvolvimento de soluções com capacidade para garantir a redução do erro na tomada de decisão bem como as que tornem possível o acompanhamento do desempenho de toda a instalação em detalhe e em tempo real são áreas nas quais se tem e se vai continuar a investir. É por isso, realmente importante, perceber onde e porquê se consome mais e é um desafio alterar esse panorama e continuar a realizar a monitorização dos registos. Para tal, a empresa Schneider Electric tem instaladas estações de medida, mais concretamente analisadores de energia, nos seus quadros elétricos, de forma a conseguir percecionar os consumos de forma mais detalhada e direcionada, ou seja, permitindo visualizar dados de todas as frações do piso. 281 Esta dissertação tem como objetivo a avaliação dos consumos de eletricidade de forma detalhada, tendo como objeto de estudo o edifício onde está sediada a empresa Schneider Eletric, de forma detalhada e a posterior identificação de hábitos ou fatores que influenciem os mesmos da forma não pretendida. Tem ainda o objetivo de avaliar o comportamento da técnica solar passiva utilizada no referido edifício. Desta forma será possível saber, não só o perfil de consumos e respetivos impactos bem como sugerir possíveis ajustes que levem a uma maior eficiência energética. Os resultados deste estudo deverão permitir às pessoas responsáveis pela entidade tomar decisões assentes em informação concreta e detalhada.

Numerical prediction of diffusion and electric field-induced iron nanoparticle transport

Zero valent iron nanoparticles (nZVI) are considered very promising for the remediation of contaminated soils and groundwaters. However, an important issue related to their limited mobility remains unsolved. Direct current can be used to enhance the nanoparticles transport, based on the same principles of electrokinetic remediation. In this work, a generalized physicochemical model was developed and solved numerically to describe the nZVI transport through porous media under electric field, and with different electrolytes (with different ionic strengths). The model consists of the Nernst–Planck coupled system of equations, which accounts for the mass balance of ionic species in a fluid medium, when both the diffusion and electromigration of the ions are considered. The diffusion and electrophoretic transport of the negatively charged nZVI particles were also considered in the system. The contribution of electroosmotic flow to the overall mass transport was included in the model for all cases. The nZVI effective mobility values in the porous medium are very low (10−7–10−4 cm2 V−1 s−1), due to the counterbalance between the positive electroosmotic flow and the electrophoretic transport of the negatively charged nanoparticles. The higher the nZVI concentration is in the matrix, the higher the aggregation; therefore, low concentration of nZVI suspensions must be used for successful field application.

Inhibition of SNF1-related protein kinase1 by trehalose 6-phosphate and other metabolites and the interrelation with plant gorwth

All life forms need to monitor carbon and energy availability to survive and this is especially true for plants which must integrate unavoidable environmental conditions with metabolism for cellular homeostasis maintenance. Sugars, in the heart of metabolism, are now recognized as crucial signaling molecules that translate those conditions. One such signal is trehalose 6- phosphate (T6P), a phosphorylated dimer of glucose molecules which levels correlate well with those of sucrose (Suc). Central integrators of stress and energy regulation include the conserved plant Snf1-related kinase1 (SnRK1) which respond to low cellular energy levels by up-regulating energy conserving and catabolic metabolism and down-regulating energy consuming processes. In 2009 T6P was shown to inhibit SnRK1. The in vitro inhibition of SnRK1 by T6P was confirmed in vivo through the observation that genes normally induced by SnRK1 were repressed by T6P and vice-versa, promoting growth processes. These observations provided a model for the regulation of growth by sugar.(...)

Reporting Income Taxes in the Banking Sector In Angola - Accounting, Tax Regulation and Financial Reporting Evidences

This research provides an insight into income taxes reporting in Angola, based on hand collected data from the annual reports of banks. Empirical studies on Angolan companies are scarce, in part due to the limited access to data. The results show that income taxes’ reporting has improved over the years 2010-2013, becoming more reliable and understandable. The Angolan Government is boosting the economic growth through tax benefits in the investment in public debt, which cause a reduction in the banks’ effective tax rate. The new income tax law will reduce the statutory tax rate from 2015 onwards and change the taxable income, resulting in shifting the focus to promoting private investment.

The cellular basis of a congenital heart defect Drosophila

RESUMO: Mutações em genes envolvidos na formação do coração e anomalias em qualquer etapa deste processo causam frequentemente malformações cardíacas, que representam o tipo mais comum de defeitos em neonatais, afetando cerca de 1% dos nascimentos por ano. Assim, estima-se que 20 milhões de pessoas sejam portadoras de um defeito cardíaco congénito. O coração da Drosophila melanogaster (mosca-da-fruta), denominado vaso dorsal, é um órgão relativamente simples que actua como uma bomba muscular, contraindo automaticamente para permitir a circulação da hemolinfa através do corpo. A formação do vaso dorsal na mosca é muito semelhante ao desenvolvimento do coração em vertebrados, representando por isso, um poderoso modelo para estudar a rede de genes e os padrões regulatórios relacionados com o desenvolvimento deste órgão. Anteriormente, nós identificámos um gene em Drosophila, darhgef10, fortemente expresso no coração em desenvolvimento e cuja deleção induz anormalidades cardíacas subtis mas prevalentes. Os mutantes para darhgef10 são viáveis e férteis no ambiente controlado de laboratório. Este trabalho teve como objectivos caracterizar fenotipicamente os mutantes nulos para darhgef10, determinar a localização subcelular da proteína dArhgef10 e investigar a base celular subjacente ao defeito no alinhamento dos cardioblastos observado nos mutantes. Os nossos resultados revelaram que a deleção de darhgef10 provoca uma severa redução da viabilidade, sem no entanto comprometer o tempo de desenvolvimento e a longevidade. Por outro lado, o aumento da expressão de darhgef10 em músculos, glândulas salivares e no disco imaginal do olho afeta drasticamente a integridade destes tecidos. A expressão ectópica de darhgef10 in vitro e in vivo revelou que a proteína está localiza no citoplasma com enriquecimento junto à membrana celular, com associação à actina F. Live imaging de embriões mutantes para darhgef10 revelou que os defeitos observados no coração podem estar associados a um defeito na adesão dos músculos alary e/ou das células pericardiais ao vaso dorsal. O homólogo humano de darhgef10, ARHGEF10, também é expresso no coração e está associação a uma maior susceptibilidade para a ocorrência de acidentes vasculares cerebrais aterotrombóticos, sugerindo que o que aprendemos sobre darhgef10 em Drosophila pode ter implicações do ponto de vista clínico para a saúde humana. ----------------------------- ABSTRACT: Mutations in genes controlling heart development and abnormalities in any of its steps frequently cause cardiac malformations, the most common type of birth defects in humans, affecting nearly 1% of births per year. Hence around 20 million adults are expected to live with a congenital heart defect. The Drosophila melanogaster heart, called dorsal vessel, is a relatively simple organ that acts as a muscular pump contracting automatically to allow the circulation of hemolymph. Drosophila heart formation shares many similarities with heart development in vertebrates providing a powerful system to study gene networks and regulatory pathways involved in heart development. We have previously identified a Drosophila gene, darhgef10, which is strongly expressed in the developing heart and when deleted, leads to flies with highly prevalent yet subtle heart abnormalities, compatible with unchallenged life in the laboratory. Our aims were to phenotypically characterize homozygous null darhgef10 mutants, characterize the subcellular localization of dArhgef10 and to study the cellular basis of the misaligned cardioblasts defect. We found that about half of darhgef10 mutants die during development. However, the survivors surprisingly have a nearly normal developmental time, adult locomotor behavior and total lifespan. Detection of transgene-derived dArhgef10 protein in vitro and in vivo using custom antibodies revealed a cytosolic protein slightly enriched in the cellular membranes and associated with F-actin. Tissue-specific darhgef10 expression disrupts the normal morphology of developing muscles, salivary glands and the eye. Live imaging of darhgef10 mutant embryos revealed that heart defect could be caused by a reduced capacity of attachment of pericardial cells and/or alary muscle to dorsal vessel. The human homolog of darhgef10 is also expressed in the heart and is a susceptibility gene for atherothrombotic stroke, suggesting that what we learn about the function of this gene and its phenotypes in Drosophila could have implications to human health.

The role of Hedgehog signaling during zebrafish larvae fin fold regeneration

Several studies have demonstrated that although the structure of the adult and larval zebrafish caudal fin is different, there are similarities at the cellular and molecular level that turn larval zebrafish fin fold a useful model to study the basic principles of regeneration. In this process, while the essential role for Hedgehog (Hh) signaling is well established in the adult zebrafish caudal fin system, its involvement in juvenile tissue regeneration is still unknown. The aim of this Master thesis was therefore to evaluate the contribution of the Hh signaling pathway to the larval zebrafish fin fold regeneration process. Accordingly, we analyzed the expression of several Hh signaling components through in situ hybridization. Here, we showed that several of these genes are effectively expressed in the larval regenerating fin tissue, suggesting a role for Hh signaling also during larval regeneration. However, divergence in the regulation of few Hh signaling components appears to exist between the adult and larval zebrafish fin regeneration processes. Nevertheless, similarly to adult caudal fin regeneration, when Hh signaling was blocked, by using cyclopamine, the larval fin fold regenerative outgrowth is severely impaired. Since larval zebrafish fin fold is ciliated, and primary cilia are closely related to Hh signaling regulation in vertebrate systems, we further addressed the role of primary cilia during larval fin fold regeneration process. To this end, we used the zebrafish iguana mutant, in which primary cilia are not formed, to study the modulation of Hh signaling expression during larval fin fold regeneration in the absence of primary cilia. Here, we found that several genes were expressed with a delay, coincident with the delay in the mutant fin fold regeneration observed in previous work. We show that Hh signaling in the fin fold is crucial to promote cell proliferation. When Hh signaling is blocked using cyclopamine there is a strong blockage of cell proliferation and regeneration is also blocked. Surprisingly, in iguana mutants where Hh signaling is impaired but not totally blocked, cell proliferation is not detected but regeneration still occurs. This raises the question about the requirement of cell proliferation in larvae fin fold regeneration. By blocking the cell cycle using aphidicolin we demonstrate that cell proliferation is not necessary for zebrafish larvae fin fold regeneration.

Synthesis of precursors of the rare 3-O-methylmannose polysaccharides present in Nontuberculous Mycobacteria

3-O-methylmannose polysaccharides (MMPs) are cytoplasmic carbohydrates synthesized by mycobacteria, which play important intracellular roles, such as for example in metabolism regulation. An important way to confirm if the inhibition of the synthesis of these polysaccharides will critically affect the survival of mycobacteria is the study of the biosynthetic pathways from these molecules on these microorganisms. The purpose of this work is the efficient synthesis of three saccharides, which are rare cellular precursors from the biosynthesis of the mycobacterial polysaccharides, allowing its study. In order to obtain these molecules, a chemical strategy to connect two precursors was used. This process is called chemical glycosylation and its importance will be highlighted as an important alternative to enzymatic glycosylation. The first objective was the synthesis of the disaccharides Methyl (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-α-D-mannopyranoside and (3-O-Methyl-α-D-mannopyra- nosyl)-(1→4)-3-O-methyl-(α/β)-D-mannopyranose. The mannose precursors were prepared before the glycosylation reaction. The same mannosyl donor was used in the preparation of both molecules and its efficient synthesis was achieved using a 8 step synthetic route from D-mannose. A different mannosyl acceptor was used in the synthesis of each disaccharide and their syntheses were also efficient, the first one a 4 step synthetic route from α-methyl-D-mannose and the second one as an intermediate from the synthesis of the mannosyl donor. The stereoselective preparation of these disaccharides was performed successfully. The second and last objective of the proposed work was the synthesis of the tetrasaccharide methyl (3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyra- nosyl-(1→4)-3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyranoside. The disaccharide acceptor and donor to be linked through a stereoselective glycosidic reaction had to be first synthesized. Several synthetic strategies were studied. Neither the precursors nor the tetrasaccharide were synthesized, but a final promising synthetic route for its preparation has been proposed.

Staying under the radar - the multifunctional LANA Protein

Many viruses have developed numerous strategies to recruit and take advantage of cellular protein degradation pathways to evade the cellular viral immune system. One such virus is the Kaposi´s Sarcoma associated herpesvirus (KSHV), first discovered in Kaposi´s Sarcoma lesions found in AIDS patients. Latency-Associated Nuclear Antigen (LANA) is a KSHV multifunctional protein responsible for tethering viral DNA to the chromosome ensuring maintenance and segregation of the viral genome during cell division. Besides its main role of viral maintenance, LANA also physically interacts with several host proteins to modulate cell functions. One such function is to recruit the EC5S ubiquitin-ligase complex by interacting with Elongin BC complex and Cullin 5 protein, which in turn ubiquitinate substrates such as NF-κB and p53 to allow persistent viral infection. Like any other post-translation modifications, ubiquitination is reversible through deubiquitination enzymes (DUBs). LANA also interacts with ubiquitin specific protease 7 (USP7), a deubiquitination enzyme involved in regulation of several proteins including p53. Interaction with USP7 is made through a conserved peptide motif, which is also present in LANA. This work addresses the role of LANA in the recruitment and modulation of the ubiquitination and deubiquitination pathways. Despite the continued efforts in uncovering new LANA interacting partners to form a functional EC5S ubiquitin-ligase complex, only MHV-68 LANA interacted directly with Elongin BC, other interactions were not direct and may require a linker protein. On the other hand, LANA interaction with USP7 was able to be analysed by X-ray structure determination. In addition to a conserved P/AxxS motif, a novel Glutamine (Gln) residue from KSHV LANA was shown to make a specific interaction with USP7. This Gln residue is also present in other herpesvirus protein and hence it might be a conserved motif within herpesviruses.