ComN (YrzD) is a small, 98-amino-acid protein recently shown to be involved in the posttranscriptional control of the late competence comE operon in Bacillus subtilis. We show here that ComN localizes to the division site and cell poles in a DivIVA-dependent fashion. Yeast two-hybrid and glutathione S-transferase pulldown experiments showed that ComN interacts directly with DivIVA. ComN is not essential for the polar assembly of the core competence DNA uptake machinery. Nevertheless, polar localization of ComN should play some role in competence acquisition because delocalization of ComN leads to a small reduction in competence efficiency. We found that ComN promotes the accumulation of its target comE mRNA to septal and polar sites. Thus, we speculate that localized translation of ComE proteins may be required for efficient competence development. Our results underscore the versatility of DivIVA as a promoter of the differentiation of bacterial poles and demonstrate that the repertoire of polarly localized molecules in B. subtilis is broad, including a regulator of gene expression and its target mRNA. Moreover, our findings suggest that mRNA localization may play a role in the subcellular organization of bacteria.

Mitochondria play a key role in adaptation during stressing situations. Cardiolipin, the main anionic phospholipid in mitochondrial membranes, is expected to be a determinant in this adaptive mechanism since it modulates the activity of most membrane proteins. Here, we used Saccharomyces cerevisiae subjected to conditions that affect mitochondrial metabolism as a model to determine the possible role of cardiolipin in stress adaptation. Interestingly, we found that thermal stress promotes a 30% increase in the cardiolipin content and modifies the physical state of mitochondrial membranes. These changes have effects on mtDNA stability, adapting cells to thermal stress. Conversely, this effect is cardiolipin-dependent since a cardiolipin synthase-null mutant strain is unable to adapt to thermal stress as observed by a 60% increase of cells lacking mtDNA (ρ0). Interestingly, we found that the loss of cardiolipin specifically affects the segregation of mtDNA to daughter cells, leading to a respiratory deficient phenotype after replication. We also provide evidence that mtDNA physically interacts with cardiolipin both in S. cerevisiae and in mammalian mitochondria. Overall, our results demonstrate that the mitochondrial lipid cardiolipin is a key determinant in the maintenance of mtDNA stability and segregation.

Background Members of the family of NEK protein kinases (NIMA-related kinases) were described to have crucial roles in regulating different aspects of the cell cycle. NEK10 was reported to take part in the maintenance of the G2/M checkpoint after exposure to ultraviolet light. NEK1, NEK5, NEK2 and NEK4 proteins on the other hand have been linked to mitochondrial functions. Methods HEK293T cells were transfected with FLAG empty vector or FLAG-NEK10 and treated or not with Zeocin. For proteomic analysis, proteins co-precipitated with the FLAG constructs were digested by trypsin, and then analyzed via LC-MS/MS. Proteomic data retrieved were next submitted to Integrated Interactome System analysis and differentially expressed proteins were attributed to Gene Ontology biological processes and assembled in protein networks by Cytoscape. For functional, cellular and molecular analyses two stable Nek10 silenced HeLa cell clones were established. Results Here, we discovered the following possible new NEK10 protein interactors, related to mitochondrial functions: SIRT3, ATAD3A, ATAD3B, and OAT. After zeocin treatment, the spectrum of mitochondrial interactors increased by the proteins: FKBP4, TXN, PFDN2, ATAD3B, MRPL12, ATP5J, DUT, YWHAE, CS, SIRT3, HSPA9, PDHB, GLUD1, DDX3X, and APEX1. We confirmed the interaction of NEK10 and GLUD1 by proximity ligation assay and confocal microscopy. Furthermore, we demonstrated that NEK10-depleted cells showed more fragmented mitochondria compared to the control cells. The knock down of NEK10 resulted further in changes in mitochondrial reactive oxygen species (ROS) levels, decreased citrate synthase activity, and culminated in inhibition of mitochondrial respiration, affecting particularly ATP-linked oxygen consumption rate and spare capacity. NEK10 depletion also decreased the ratio of mtDNA amplification, possibly due to DNA damage. However, the total mtDNA content increased, suggesting that NEK10 may be involved in the control of mtDNA content. Conclusions Taken together these data place NEK10 as a novel regulatory player in mitochondrial homeostasis and energy metabolism.

Resumo:As monitorias têm sido atualmente muito utilizadas pelas universidades como processos auxiliares de ensino, nos quais alunos são selecionados para ajudar os professores no desenvolvimento e aperfeiçoamento de algumas das atividades técnico-didáticas, proporcionando assim mais um recurso para o acompanhamento dos alunos no processo de aprendizagem. Objetivou-se com esse trabalho analisar a contribuição exercida pelas monitorias de Bioquímica na aprendizagem dessa disciplina nos alunos da área da saúde do Unileste-MG, sob o ponto de vista de alunos, monitores e professores. Os dados foram obtidos através de questionários aplicados aos alunos e entrevistas semi-estruturadas realizadas com monitores e professores. Observou-se que grande porcentagem de alunos apresentam dificuldades no entendimento da disciplina, e que a maior porcentagem destes freqüentam as monitorias somente nos dias anteriores às provas. Porém, declaram que elas exercem contribuição para a aprendizagem da disciplina. Os monitores e professores concordam que as monitorias contribuem para a aprendizagem. No entanto, ressaltam a importância da freqüência regular dos alunos às monitorias, assim como maior participação em sala de aula. As monitorias também constituem estímulo aos monitores para inserção em outras atividades, tais como projetos de extensão, docência e pesquisa. Abstract:Teachers assistants (TA) have been largely employed at universities to help teachers in educational procedures. TAs are selected among graduate students and work on the development and improvement of some technician-didactic activities, providing also extra support for the undergraduates in their learning process. The project here described has analyzed the contribution of TAs in Biochemistry courses for Health Sciences students at Unileste-MG. Evaluations were made by either students, teachers and TAs. The data was collected through questionnaires filled in by the students and semi-structured interviews with students, TAs and teachers. The results show that a high percentage of the students present difficulties in the understanding of the contents. Nevertheless, they ask for TAs help only a few days before the written tests are due. However, they still declare that TAs contribute for the learning of biochemistry. TAs and teachers share their opinion but point out the importance of a more constant support by TAs and a more intense participation in the classroom as well. By acting as TAs, graduate students are stimulated by their insertion in other academic activities such as teaching, research and extension projects.Endereço para contato:

Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal of bulky, helix-distorting DNA lesions, like ultraviolet damage or cisplatin adducts, but its role in the repair of lesions generated by oxidative stress is still not clear. The helicase XPD/ERCC2, one of the two helicases of the transcription complex IIH, together with XPB, participates both in NER and in RNA pol II-driven transcription. In this work, we investigated the responses of distinct XPD-mutated cell lines to the oxidative stress generated by photoactivated methylene blue (MB) and KBrO3 treatments. The studied cells are derived from patients with XPD mutations but expressing different clinical phenotypes, including xeroderma pigmentosum (XP), XP and Cockayne syndrome (XP-D/CS) and trichothiodystrophy (TTD). We show by different approaches that all XPD-mutated cell lines tested were sensitive to oxidative stress, with those from TTD patients being the most sensitive. Host cell reactivation (HCR) assays showed that XP-D/CS and TTD cells have severely impaired repair capacity of oxidised lesions in plasmid DNA, and alkaline comet assays demonstrated the induction of significantly higher amounts of DNA strand breaks after treatment with photoactivated MB in these cells compared to wild-type cells. All XPD-mutated cells presented strong S/G2 arrest and persistent γ-H2AX staining after photoactivated MB treatment. Taken together, these results indicate that XPD participates in the repair of lesions induced by the redox process, and that XPD mutations lead to differences in the response to oxidatively induced damage.

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pelo carinho, amor e ensinamentos que formam meus valores como pessoa. Ao Carlos R. Prudêncio pela paciência, convivência, amor, dedicação, apoio em diversos momentos, incluindo madrugadas de leitura de tese, e sobretudo me incentivando a sempre olhar as coisas pelo lado positivo. Aos meus amigos em especial João, Paulo Tonolli, Gleidson, Adjací, Serginho, Ailton. Obrigada pelo carinho e apoio ao longo dessa caminhada. À Adriana M. P. Wendel pela ajuda técnica, conselhos, orientações e conversas sempre agradáveis. À Andressa P. Costa pelo carinho e também pelo auxílio técnico a amizade sempre presente. À Juliane Raveli ajuda técnica, conselhos, amizade, risadas e conversas sempre agradáveis. Aos colegas de departamento de diversos laboratórios que gentilmente cederam espaço em seus laboratórios, me ensinaram a usar equipamentos, disponibilizaram reagentes e até mesmo me ouviram em momentos diversos enquanto tomávamos café. Ao pessoal da secretaria de pós-graduação pelo carinho no atendimento e por toda ajuda que prestam sempre quando solicitados. A todas as pessoas que contribuíram de alguma forma para o meu amadurecimento, me ensinando valiosas lições de vida.