Molecular regulation and analysis of neural stem and cancer cell characteristics

Every single cell within an organism is constantly facing the demanding task of preserving the integrity of its genomic material to secure proper function and clearance from disease. To accomplish this cells rely on DNA repair mechanisms that are well-conserved throughout evolution and highly specialized in removing particular types of DNA damage. The ability of rapidly repairing DNA lesions is paramount for cells in both the developing and adult central nervous system (CNS), not only to preserve their differentiation potential but also to ensure that post mitotic cells, which comprise the CNS vast majority, are spared and continue to contribute to the maintenance of CNS homeostasis. Due to their putative enhanced repair capacity, neural stem cells (NSCs) may not only play a critical role in sustaining the pool of neural progenitors over the course of neurogenesis, but also in certain conditions, replenish the loss of terminally differentiated cells due to a variety of damage inducing events. The work presented in this thesis aimed to further explore the significance of DNA glycosylase activity both in neural stem and cancer cells and to develop a probe for rapid assessment of neuronal characteristics in neurons differentiated from NSCs. In paper I we propose the DNA glycosylases OGG1 and NEIL3 play roles beyond direct removal of oxidized bases and that they are necessary for typical expression levels of genes conferring normal neural stem cell characteristics and multipotency required for differentiation into the various cell lineages found in the mammalian CNS. Further, we show that NEIL3 deficiency can enhance formation of senescence related heterochromatin foci, suggesting an influence in regulatory functions of this cellular pathway. In paper II we first demonstrate we can generate C6 glioma cells with stem cell-like characteristics (C6SCs), as it could be assessed by an increased ability of these cells to differentiate into astrocytes and neurons after CNTF and VPA treatment respectively. In addition, we show that RNA knockdown of the DNA glycosylase OGG1 in C6SCs affects differentiation potential and increases the histone modification mark, acetylation of histone 3 in lysine 56 (H3K56ac) associated with increased DNA damage response (DDR). This enhancement of the DDR may confer a certain degree of resistance to cancer therapies that should be carefully taken in account. Finally, in paper III we introduce a new method for the use of a commercially available voltage sensitive dye (VSD), JPW3027, for accurate characterization of neurons differentiated from NCSs, regarding their ability to generate action potentials (AP). We found that extracellular application of this dye improves labeling of cellular processes, and upon excitation it reports changes in fluorescent translating precise AP kinetics, which are of overall superior quality compared to calcium indicators. Furthermore, JPW3027 proved to possess a lesser degree of toxicity, which represents a great advantage when monitoring cells for extended periods of time after dye loading. Finally, we propose the use of a finite element model of the NSC culture cover slip to optimize electrode positioning relatively to the patched cells this way producing an electrical stimulation that is homogenous to all cells. With this approach we are able to predict isopotential fields where electrodes can be placed minimizing the perturbation of cells away from the field of view

Pitaia: perspetivas e dificuldades de uma “nova” cultura

No Algarve, onde existem condições edafoclimáticas favoráveis ao cultivo de algumas espécies frutícolas exóticas, oriundas de climas tropicais ou subtropicais, surge o interesse no cultivo da pitaia. Esta cultura apresenta altas produções em alguns países e é adequada para terrenos de pequenas dimensões. Ela acaba por satisfazer também a necessidade de diversificar a fruticultura da região. Devido à sua aparência exótica e ao seu alto valor nutritivo, a pitaia tem sido cada vez mais procurada em diversos países, principalmente pelos mercados asiáticos e europeus. Para desenvolver a cultura da pitaia no Algarve foi constituído um grupo operacional cujo objetivo principal visa inovar ao nível das tecnologias de produção para a pitaia vermelha, testando a sua produtividade, rusticidade e qualidade dos frutos. Começou-se por fazer um levantamento da situação desta cultura em Portugal. Estão também a ser instalados vários campos de ensaio/recolha de dados, sobre várias espécies de pitaia vermelha.info:eu-repo/semantics/publishedVersio

A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures

BACKGROUND: Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca(2+)-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. METHODOLOGY/PRINCIPAL FINDINGS: In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. CONCLUSIONS/SIGNIFICANCE: Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture

A poda de manutenção em citrinos

A poda em citricultura é muitas vezes tema de discórdia: enquanto uns a consideram uma prática essencial ou mesmo indispensável, outros acham que é uma prática desnecessária, cujos benefícios não compensam os custos. Na realidade, se realizada da forma e na época adequada, a poda pode funcionar como estratégia para melhorar a produtividade e qualidade da produção, além de atenuar vários problemas da cultura dos citrinos.info:eu-repo/semantics/publishedVersio

The implementation of radiation technology program in Portugal

The development of ionizing radiation pplications for Industrial purposes in Portugal began near of 1982 with the support of IAEA. The main steps to put forward prior to the implementation were the sitting and the design study in order to build up the facility. Subsequently, the main parameters to be achieved were the construction, the commissioning, the operation, the maintenance and the foreseen decommission. Once a quality system for the gamma facility was established, the following stage is to develop, validate and control the terilization/disinfection process. The research activities carried out in the UTR have been closely related with the main applications of this technology namely, the sterilization of medical devices and pharmaceuticals and other products’ decontamination. Recently, a research Cobalt-60 equipment was upgraded and a LINAC was implemented in order to sustain the R&D. Fundamental and development research is ngoing in order to understand the irradiation mechanisms of action and to apply the technology with safety and quality patterns.The first author would like to thank Gulbenkian Foundation in Portugal and NIC2010 the financial support for the opportunity to participate at NAARRI International Conference

Dimeric Self-assembling via Hydrogen Bonding and Emissive Behavior of a New Copper (I) Complex

This work describes the synthesis, structural characterization and emissive behavior of a new copper (I) complex based on 1-thiocarbamoyl-5-(4-methoxiphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole ligand. A dimeric self-assembling via hydrogen bonding was determined by analyzing the short contacts present in the solid-state structure by means of X-ray crystallography. The spectroscopic properties were determined using UV-Vis and fluorescence experiments and an interesting behavior as bluish luminescence was assigned mainly to the mixed (MLCT + IL) electronic transitions of the Cu(I)d10 ® (S=C–Nligand) type. The complete characterization of the new copper (I) complex also included elemental analyses and IR spectroscopy. DOI: http://dx.doi.org/10.17807/orbital.v9i1.95

Reactivation of latent HIV-1 in vitro using an ethanolic extract from Euphorbia umbellata (Euphorbiaceae) latex

Euphorbia umbellata (E. umbellata) belongs to Euphorbiaceae family, popularly known as Janauba, and its latex contains a combination of phorbol esters with biological activities described to different cellular protein kinase C (PKC) isoforms. Here, we identified deoxi-phorbol esters present in E. umbellata latex alcoholic extract that are able to increase HIV transcription and reactivate virus from latency models. This activity is probably mediated by NF-kB activation followed by nuclear translocation and binding to the HIV LTR promoter. In addition, E. umbellata latex extract induced the production of pro inflammatory cytokines in vitro in human PBMC cultures. This latex extract also activates latent virus in human PBMCs isolated from HIV positive patients as well as latent SIV in non-human primate primary CD4+ T lymphocytes. Together, these results indicate that the phorbol esters present in E. umbellata latex are promising candidate compounds for future clinical trials for shock and kill therapies to promote HIV cure and eradication.Research and experimental expenses were funded by the grant E26/2015064289 from FAPERJinfo:eu-repo/semantics/publishedVersio