The ROS wheel: refining ROS transcriptional footprints

In the last decade, microarray studies have delivered extensive inventories of transcriptome-wide changes in messenger RNA levels provoked by various types of oxidative stress in Arabidopsis (Arabidopsis thaliana). Previous cross-study comparisons indicated how different types of reactive oxygen species (ROS) and their subcellular accumulation sites are able to reshape the transcriptome in specific manners. However, these analyses often employed simplistic statistical frameworks that are not compatible with large-scale analyses. Here, we reanalyzed a total of 79 Affymetrix ATH1 microarray studies of redox homeostasis perturbation experiments. To create hierarchy in such a high number of transcriptomic data sets, all transcriptional profiles were clustered on the overlap extent of their differentially expressed transcripts. Subsequently, meta-analysis determined a single magnitude of differential expression across studies and identified common transcriptional footprints per cluster. The resulting transcriptional footprints revealed the regulation of various metabolic pathways and gene families. The RESPIRATORY BURST OXIDASE HOMOLOG F-mediated respiratory burst had a major impact and was a converging point among several studies. Conversely, the timing of the oxidative stress response was a determining factor in shaping different transcriptome footprints. Our study emphasizes the need to interpret transcriptomic data sets in a systematic context, where initial, specific stress triggers can converge to common, aspecific transcriptional changes. We believe that these refined transcriptional footprints provide a valuable resource for assessing the involvement of ROS in biological processes in plants

Plant proteases and programmed cell death

Proteolysis affects many processes in plant development and during stress responses, as well as being crucial in cellular protein homeostasis and recycling of resources. Beyond bulk degradation, proteases can have important signaling functions or affect cellular pathways by precise cleavage of signaling proteins. This special issue covers key research themes in the diverse but increasingly interconnected fields of programmed cell death (PCD) and plant protease activity. Future trends are also highlighted, such as accelerated substrate discovery facilitated by large-scale deposition of N-terminomic data to easily accessible databases, or better profiling using genetically encoded protease activity reporters

Discovery of organellar calcium signaling components in Arabidopsis thaliana

In dieser Dissertation wird die Rolle von Organellen in der Kalzium-abhängigen Signaltransduktion in Pflanzen untersucht. Pflanzen und andere eukaryotische Organismen nutzen Kalzium als sekundären Botenstoff („secondary messenger“), um sich schnell an veränderte Umweltbedingungen anzupassen, oder um ihre Entwicklungsprozesse zu steuern. Die Bedeutung von Kalzium-abhängiger Signaltransduktion („calcium signaling“) in der Pflanzenphysiologie ist heute gut etabliert, ist aber bisher hauptsächlich auf zytosolische Prozesse fokussiert. Um weitere Einblicke in „calcium signaling“ in pflanzlichen Organellen zu bekommen, habe ich den Einfluss von Kalzium auf Plastiden, Mitochondrien und Peroxisomen untersucht, und zum Teil auch den Einfluss dieser Organellen auf das „calcium signaling“ im Rest der Zelle. Ich habe meine Arbeit mit einer Suche nach Kalzium-bindenden Proteinen in Chloroplasten in einem gerichteten proteomischen Ansatz und intensiven Recherchen in Datenbanken begonnen. Am Ende waren die Resultate dieser Arbeit nicht nur auf Plastiden beschränkt, da ich auch neuartige Komponenten des „calcium signaling“ in Mitochondrien und Peroxisomen entdeckt habe. Die Proteomischen Ansätze führten zur Identifizierung von zwei bestätigten Kalzium-bindenden Proteinen im Chloroplasten: LENA (Little E-enriched protein A), welches vermutlich eine Komponente des lange gesuchten Kalziumspeichers im Plastiden darstellt, und SAMTL (S adenosyl methionine transporter-like), einem Mitglied der Mitochondrial Carrier Familie (MCF), welches vermutlich in einer Kalzium-abhängigen Weise SAM in Chloroplasten transportiert. Zusätzlich wurden drei weitere Kalzium-abhängige MCF Proteine identifiziert, die ATP/Phosphat Carrier APC1, 2 und 3. Phylogenetische Analysen und funktionelle Komplementation von Hefemutanten legen nahe, dass APC1-3 in Mitochondrien lokalisiert sind und eine Rolle unter Sauerstoffmangelbedingungen spielen könnten. Außerdem wurde ein organellenlokalisiertes EFHand Protein mit einer Masse von 18 kDa (OEF18) identifiziert, welches dual im äußeren Chloroplasten Envelope und in Peroxisomen lokalisiert ist und in den Prozess der Organellenteilung involviert sein lönnte. Zusätzlich zu meinen Ansätzen, neuartige organellenlokalisierte Kalzium-bindende Proteine zu identifizieren, habe ich auch einen möglichen „cross-talk“ zwischen „calcium signaling“ und Proteinphosphorylierung im Chloroplasten untersucht. Dabei konnte eindeutig eine Kalziumabhängige Phosphorylierung der drei Chloroplastenproteine, PsaN (Photosystem I Untereinheit N), der FtsH Protease VAR1 (Variegated 1) und CAS (Calcium Sensor Protein) gezeigt werden. Über die funktionelle Bedeutung dieser neuen Regulation kann soweit nur spekuliert werden, aber diese Ergebnisse bilden jetzt eine stabile Basis für weitere Untersuchungen über die Rolle der Kalziumabhängigen Protein Phosphorylierung in Chloroplasten.My thesis is focused on the role of organelles in plant calcium signaling. Plants and other eukaryotic organisms use calcium ions as secondary messengers in order to acclimate rapidly to changes of the environment or to assist in developmental programs. The importance of calcium signaling on plant physiology is well established today, but has so far focused mainly on calcium signaling in the cytosol. To get insights into plant organellar calcium signaling, I have studied the impact of calcium on plastids, mitochondria and peroxisomes, and to a minor extent also the influence of these organelles on calcium signaling in the rest of the cell. Initially, I started with a search for calcium binding proteins in the chloroplast by targeted proteomics and data mining. However, the results of this work were in the end not only restricted to plastids as I discovered novel calcium signaling components of the mitochondria and peroxisomes. The proteomic approaches resulted in the identification of two confirmed chloroplast calcium binding proteins. LENA (Little E-enriched protein A) is predicted to be a protein component of the long-hypothesized calcium storage of the plastid. SAMTL (S-adenosyl methionine transporter-like) is a member of the mitochondrial carrier family and predicted to transport SAM into chloroplasts in a calcium-dependent manner. The three calcium-dependent mitochondrial carrier family (MCF) proteins, APC1, 2 and 3 (ATP/phosphate carriers 1, 2 and 3) localize to mitochondria. Phylogenetic analysis and complementation of yeast mutants suggested APC1, 2 and 3 to be mitochondrial ATP importers, which could be needed during periods of anoxia. Furthermore an organellar EF-hand protein of 18 kDa (OEF18) was discovered in the course of a study on the impact of protein N-acylation for organellar targeting. This protein was identified by data mining as well as in the proteomic approach. Subsequent work showed that OEF18 is dually targeted to the plastid outer envelope and peroxisomal membrane and might be involved in the process of organellar division. In addition to my approaches to identify novel organellar Ca2+ binding proteins, I have studied a potential crosstalk between calcium signaling and protein phosphorylation in chloroplasts. Three chloroplast proteins, PsaN (subunit N of Photosystem I), the FstH protease VAR1 (Variegated 1) and CAS (Calcium sensing protein) were conclusively found to be phosphorylated in a calcium dependent manner. The implications of this novel regulation are speculative, but the presented findings provide a stable base to further investigate the impact of calcium dependent protein phosphorylation on chloroplast function

N-terminal proteomics assisted profiling of the unexplored translation initiation landscape in Arabidopsis thaliana

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well-and poorly-annotated genomes

Saúde vocal do professor em sua atuação com crianças de desenvolvimento atípico

Monografia (especialização)—Universidade de Brasília, Instituto de Psicologia, Departamento de Psicologia Escolar e do Desenvolvimento Humano, Programa de Pós-Graduação em Processos de Desenvolvimento Humano e Saúde, 2015.A qualidade da saúde vocal, em especial dos professores, depende basicamente de se ter conhecimento de como acontece sua produção, de hábitos saudáveis como uma forma de prevenção. Nesse sentido o presente estudo tem como objetivo refletir sobre os aspectos que interferem na saúde do professor, principalmente àqueles que atuam com crianças com desenvolvimento atípico, com enfoque às alterações vocais que acometem estes profissionais. Para tanto foi realizada uma pesquisa de campo com 06 (seis) professores da rede pública que atuam em sala de aula de ensino regular e aqueles que desempenham atividades na Sala de Recursos, no Atendimento Educacional Especializado (AEE) com alunos de Desenvolvimento Atípico. Apesar disso, enfatizou-se a importância da preparação do professor para a manutenção de uma boa saúde vocal, bem como uma melhor preparação para os profissionais que atuam com crianças com necessidades educacionais especiais (NEE)

Inversores com controle automatizado operando em microrredes com cargas desequilibradas

This work aims at dimensioning and developing an automated hybrid microgrid using photovoltaic generation and an energy storage system, to be implemented at the Faculty of Engineering of the Federal University of Juiz de Fora (UFJF). Microgrids can be defined as an integrated system that involves distributed energy resources and several electrical loads operating as an autonomous network, either in parallel or isolated from the main electrical network. One of the great advantages of microgrids is the possibility of testing devices and control required for the development of smart electrical networks, with less risk and associated cost. The system was modeled and simulated in Matlab / Simulink version 2018a, with automated operations, detecting the occurrence of islanding and later doing the resynchronization process with the main network, under unbalanced load conditions. A multifunctional converter operates as Grid Forming Converter, supplying highly unbalanced loads. A converter in this scenario will need 4 arms to perform the control of the zero sequence components resulting from the load unbalance. Its operation in the grid-forming mode has not been fully addressed in the literature. The contribution of this work also includes the use of controllers based on the synchronous coordinate system, avoiding the use of complex controllers and obtaining an rapid transient response during system disturbances. Simulation results obtained were used to validate the proposed methodology, showing the functionalities of the implemented inverter, working automatically in the detection of the occurrence of islanding and resynchronization of the microgrid with the main grid. By comparing the results achieved with and without the voltage unbalance compensation algorithm, its effectiveness was shown, reducing the voltage unbalance factor (VUF) index to zero, keeping the output voltage on the PAC balancedEste trabalho visa o dimensionamento e o desenvolvimento de uma microrrede híbrida automatizada empregando geração fotovoltaica e um sistema de armazenamento de energia, a ser implementada na Faculdade de Engenharia da Universidade Federal de Juiz de Fora (UFJF). Microrredes podem ser definidas como um sistema integrado que envolve recursos de energia distribuídos e várias cargas elétricas operando como uma rede autônoma, seja em paralelo ou ilhada da rede elétrica principal. Uma das grandes vantagens de se trabalhar com microrredes está na possibilidade de testar dispositivos e controles necessários para o desenvolvimento de redes elétricas inteligentes, com menor risco e custo associado. O sistema foi modelado e simulado no Matlab/Simulink versão 2018a, com operações automatizadas, detectando a ocorrência de ilhamento e posteriormente fazendo o processo de ressincronização com a rede principal, sob condições de cargas desequilibradas. O conversor multifuncional opera como Formador de Rede, suprindo cargas altamente desequilibradas. Um conversor nesse cenário necessitará de 4 braços para realizar o controle das componentes de sequência zero advindas do desbalanço de carga e sua operação no modo formador de rede é um tema pouco abordado na literatura. Tem-se ainda como contribuição deste trabalho o fato de se utilizar controladores baseados no sistema de coordenadas síncrono, evitando a utilização de controladores complexos e obtendo uma resposta transitória rápida durante os distúrbios do sistema. Resultados de simulação digital obtidos foram utilizados para validar a metodologia proposta, mostrando as funcionalidades do inversor implementado, trabalhando automaticamente na detecção de ocorrência de ilhamento e ressincronização da microrrede com a rede principal. Por meio da comparação dos resultados alcançados, mostrou-se a eficácia do algoritmo do compensador de desequilíbrio de tensão, reduzindo o índice do fator de desequilíbrio de tensão (VUF) para zero, mantendo a tensão de saída no PAC equilibrada