Inibição de cinases e/ou fosfatases celulares por bibliotecas de siRNA:aplicação na infecção do HIV-1

Tese de mestrado, Biologia (Biologia Molecular e Genética), 2008, Universidade de Lisboa, Faculdade de CiênciasO mecanismo de reparação do DNA após a integração do HIV-1 ainda não foi esclarecido. A cinase dependente de DNA (DNA-PK) já foi descrita como podendo desempenhar um papel importante na integração, contudo existem estudos que contestam tal afirmação. Este trabalho tem como objectivo esclarecer o papel da DNA-PK na replicação do HIV-1. Neste sentido, criaram-se clones onde a subunidade catalítica da DNA-PK (DNA-PKcs) é silenciada pela expressão estável de shRNAs. O nível de silenciamento da DNA-PKcs foi avaliado por Western Blot, verificando-se uma diminuição acentuada dos níveis de expressão de DNA-PKcs para todos os clones. Através destes clones pretendeu avaliar-se a importância da DNA-PKcs para a replicação do HIV-1. Para tal, infectaram-se os clones com HIV-1 e determinou-se a sua resistência à infecção ao fim de sete dias. Todos os clones demonstraram uma inibição quase total de produção de partículas virais e da proteína da cápside (p24), demostrando que a DNA-PKcs é importante para o ciclo de replicação do HIV-1. Uma vez que a DNA-PKcs tem um papel importante na reparação de danos de DNA de cadeia dupla (DSBs), foi avaliado se a infecção com HIV-1 leva à formação de DSBs e se a DNA-PKcs está envolvida na sua reparação. Demonstrou-se que a infecção de HIV-1 leva à fomação de DSBs. Obtiveram-se alguns dados preliminares onde a formação das DSBs devido à infecção com HIV-1 não é tão evidente nos clones que expressam shRNAs contra DNA-PKcs, sugerindo que o papel da DNA-PKcs poderá não ser importante ao nível da reparação das DSBs provocadas pela infecção do HIV-1. O esclarecimento do papel que a DNA-PKcs desempenha na replicação do HIV-1 levará a um maior conhecimento acerca da dinâmica entre o vírus e o hospedeiro, o que poderá levar à descoberta de novos alvos terapêuticos eficazesThe mechanism of DNA repair after HIV-1 integration is yet to be fully understood. The DNA dependent protein kinase (DNA-PK) has been described as possibly having an important role in integration, however there are studies that contradict this statement. The goal of this work is to clarify the role of DNA-PK in HIV-1 replication. For this purpose, clones where the catalytic subunit of the DNA-PK (DNA-PKcs) is silenced by the stable expression of shRNAs were created. The knock down level of DNA-PKcs was evaluated by Western Blot, showing a considerable decrease of DNA-PKcs expression levels in every clone. These clones allow for the study of the importance of DNA-PKcs in HIV-1 replication. To this end, the clones were infected with HIV-1 and their resistance to infection was evaluated after seven days. All clones showed an almost complete inhibition of viral particle and capsid protein (p24) production, showing that DNA-PKcs is important for HIV-1 replication. Since DNA-PKcs has an important role in the repair of DNA double strand breaks (DSBs), it was evaluated if infection with HIV-1 leads to the formation of DSBs and if DNA-PKcs is involved in their repair. It was demonstrated that infection with HIV-1 leads to the formation of DSBs. Some preliminary results show that the formation of DSBs due to HIV-1 infection is not evident in the clones that have stable expression of shRNAs against DNA-PKcs, suggesting that the role of DNA-PKcs in HIV-1 replication might not be important in the repair of DSBs formed during infection. Understanding the role of DNA-PKcs in HIV-1 replication could widen our knowledge on the dynamics between the virus and the host. This could lead to the discovery of novel therapeutic targets effective in the battle against HI

O desafio da gestão do talento no contexto da transformação digital em curso - uma análise a empresas do Alentejo.

A transformação digital está a pressionar as organizações a introduzir novas tecnologias no negócio, alterando modelos de negócio e a cultura organizacional, bem como adaptar os seus trabalhadores. Com a virtualização dos processos, surge a necessidade de novos requisitos de perfis de pessoas, com qualificações e capacidades de conhecimento e habilidades. Este trabalho investiga o desafio da gestão do talento face à transformação digital, tentando compreender de que modo as empresas detetam, atraem, retêm e formam os seus colaboradores. Foi realizado um estudo de caso a duas empresas em Évora. Realizaram-se entrevistas a uma especialista em talento e aos responsáveis de Recursos Humanos de duas empresas. Da análise, destaca-se: a empresa X, com lacunas no contexto da transformação digital, encontra-se numa fase embrionária no que toca à gestão do talento. Por outro lado, a empresa Lobosolar, que acompanha a transformação digital, tem conseguido fazer a gestão do seu talento

Preliminary characterization of a Moroccan honey with a predominance of Bupleurum spinosum pollen

Honey with Bupleurum spinosum (zandaz) as a main pollen source has not been the subject of previous detailed study. Therefore, twelve Moroccan samples of this honey were subjected to melissopalynological, physicochemical and microbiological quality characterization, as well as antioxidant activity assessment. From a quality point of view, almost all samples were within the limits established by Codex Alimentarius, and/or the European legislation. All samples presented predominance of B. spinosum pollen (more than 48%). Relatively high levels of trehalose (1.3-4.0 g/100 g) and melezitose (1.5-2.8 g/100 g) were detected. Those sugars, not common in monofloral honeys, could be used as an important factor to discriminate zandaz honey. Flavonoid content correlated positively with the honey color, melanoidin and polyphenol content, and negatively with the IC50 values of scavenging ABTS (2,2' - azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radicals, while proline amount correlated negatively with IC50 values of nitric oxide scavenging activity and chelating power. This correlation supports the use of anti-oxidant activities as important variables for PCA (principal component analysis). Both components explained 70% from the given data, and showed certain homogeneity upon analyzed samples independent of the region, suggesting the importance of B. spinosum nectar in the resulting honey characteristics.Fundacao para a Ciencia e Tecnologia for Research Center [UID/BIM/04773/2013 CBMR 1334, UID/AGR/00239/2013, UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569)]; ERDF through the COMPETE - Programa Operacional Competitividade e Internacionalizacao (POCI

Primary metabolism is distinctly modulated by plant resistance inducers in Coffea arabica leaves infected by Hemileia vastattrix

Original ResearchEpidemics of coffee leaf rust (CLR) leads to great yield losses and huge depreciation of coffee marketing values, if no control measures are applied. Societal expectations of a more sustainable coffee production are increasingly imposing the replacement of fungicide treatments by alternative solutions. A protection strategy is to take advantage of the plant immune system by eliciting constitutive defenses. Based on such concept, plant resistance inducers (PRIs) have been developed. The Greenforce CuCa formulation, similarly to acibenzolar-S-methyl (ASM), shows promising results in the control of CLR (Hemileia vastatrix) in Coffea arabica cv. Mundo Novo. The molecular mechanisms of PRIs action are poorly understood. In order to contribute to its elucidation a proteomic, physiological (leaf gas-exchange) and biochemical (enzymatic) analyses were performed. Coffee leaves treated with Greenforce CuCa and ASM and inoculation with H. vastatrix were considered. Proteomics revealed that both PRIs lead to metabolic adjustments but, inducing distinct proteins. These proteins were related with photosynthesis, protein metabolism and stress responses. Greenforce CuCa increased photosynthesis and stomatal conductance, while ASM caused a decrease in these parameters. It was further observed that Greenforce CuCa reinforces the redox homeostasis of the leaf, while ASM seems to affect preferentially the secondary metabolism and the stress-related proteins. So, the PRIs prepare the plant to resist CLR but, inducing different defense mechanisms upon pathogen infection. The existence of a link between the primary metabolism and defense responses was evidenced. The identification of components of the plant primary metabolism, essential for plant growth and development that, simultaneously, participate in the plant defense responses can open new perspectives for plant breeding programsinfo:eu-repo/semantics/publishedVersio

Produtos alternativos para o controlo de doenças das plantas

Os fosfitos e os silicatos, embora considerados nutrientes minerais das plantas, podem também funcionar como produtos alternativos no controlo de doenças em diferentes culturas. Estes minerais inorgânicos podem atuar diretamente contra os microrganismos patogénicos e/ou induzir as respostas de defesa das plantasinfo:eu-repo/semantics/publishedVersio

Novel HIV-1 Knockdown Targets Identified by an Enriched Kinases/Phosphatases shRNA Library Using a Long-Term Iterative Screen in Jurkat T-Cells

HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies

Latin Americans show wide-spread Converso ancestry and imprint of local Native ancestry on physical appearance

Historical records and genetic analyses indicate that Latin Americans trace their ancestry mainly to the intermixing (admixture) of Native Americans, Europeans and Sub-Saharan Africans. Using novel haplotype-based methods, here we infer sub-continental ancestry in over 6,500 Latin Americans and evaluate the impact of regional ancestry variation on physical appearance. We find that Native American ancestry components in Latin Americans correspond geographically to the present-day genetic structure of Native groups, and that sources of non-Native ancestry, and admixture timings, match documented migratory flows. We also detect South/East Mediterranean ancestry across Latin America, probably stemming mostly from the clandestine colonial migration of Christian converts of non-European origin (Conversos). Furthermore, we find that ancestry related to highland (Central Andean) versus lowland (Mapuche) Natives is associated with variation in facial features, particularly nose morphology, and detect significant differences in allele frequencies between these groups at loci previously associated with nose morphology in this sample.Instituto Multidisciplinario de Biología Celula

Latin Americans show wide-spread Converso ancestry and imprint of local Native ancestry on physical appearance

Historical records and genetic analyses indicate that Latin Americans trace their ancestry mainly to the intermixing (admixture) of Native Americans, Europeans and Sub-Saharan Africans. Using novel haplotype-based methods, here we infer sub-continental ancestry in over 6,500 Latin Americans and evaluate the impact of regional ancestry variation on physical appearance. We find that Native American ancestry components in Latin Americans correspond geographically to the present-day genetic structure of Native groups, and that sources of non-Native ancestry, and admixture timings, match documented migratory flows. We also detect South/East Mediterranean ancestry across Latin America, probably stemming mostly from the clandestine colonial migration of Christian converts of non-European origin (Conversos). Furthermore, we find that ancestry related to highland (Central Andean) versus lowland (Mapuche) Natives is associated with variation in facial features, particularly nose morphology, and detect significant differences in allele frequencies between these groups at loci previously associated with nose morphology in this sample.Instituto Multidisciplinario de Biología Celula

All-sky search for gravitational-wave bursts in the second joint LIGO-Virgo run

We present results from a search for gravitational-wave bursts in the data collected by the LIGO and Virgo detectors between July 7, 2009 and October 20, 2010: data are analyzed when at least two of the three LIGO-Virgo detectors are in coincident operation, with a total observation time of 207 days. The analysis searches for transients of duration < 1 s over the frequency band 64-5000 Hz, without other assumptions on the signal waveform, polarization, direction or occurrence time. All identified events are consistent with the expected accidental background. We set frequentist upper limits on the rate of gravitational-wave bursts by combining this search with the previous LIGO-Virgo search on the data collected between November 2005 and October 2007. The upper limit on the rate of strong gravitational-wave bursts at the Earth is 1.3 events per year at 90% confidence. We also present upper limits on source rate density per year and Mpc^3 for sample populations of standard-candle sources. As in the previous joint run, typical sensitivities of the search in terms of the root-sum-squared strain amplitude for these waveforms lie in the range 5 10^-22 Hz^-1/2 to 1 10^-20 Hz^-1/2. The combination of the two joint runs entails the most sensitive all-sky search for generic gravitational-wave bursts and synthesizes the results achieved by the initial generation of interferometric detectors.Comment: 15 pages, 7 figures: data for plots and archived public version at https://dcc.ligo.org/cgi-bin/DocDB/ShowDocument?docid=70814&version=19, see also the public announcement at http://www.ligo.org/science/Publication-S6BurstAllSky