La dynamique et la distribution mitochondriale sont essentielles pour l’homéostasie énergétique cellulaire. CLUH est une protéine indispensable à la distribution mitochondriale, dont la déplétion provoque une agrégation mitochondriale périnucléaire. Afin de comprendre le rôle de CLUH dans le métabolisme cellulaire, nous avons généré des cellules knockout CLUH par la méthode CRISPR-cas9. Nos résultats montrent que l’agrégation mitochondriale est associée à la diminution de la taille cellulaire et à la réduction quantitative des complexes de la chaîne respiratoire, menant ainsi à des défauts de la phosphorylation oxydative. Cette déficience énergétique est due à la perturbation de la traduction mitochondriale, et provoque un shift métabolique vers la glycolyse. Le profil métabolique des cellules KO montre un dysfonctionnement du cycle de Krebs et une altération de l’oxydation des acides gras. Dans ce sens, nous avons déterminé une fonction cruciale de CLUH dans le couplage de la distribution mitochondriale au contrôle de l’état cellulaire énergétique et métabolique. Pour approfondir l’analyse de la fonction de CLUH, nous avons effectué une étude de prédiction des domaines fonctionnels in silico, et avons identifié cinq domaines évolutivement conservés au sein de la séquence primaire de CLUH. De plus, nous démontrons que CLUH oligomérise en tétramères et en octomères, qui sont déstabilisés par l’expression ectopique de formes tronquées de CLUH dépourvues des domaines Clu-Nou TPR, par un effet dominant négatif. En résumé, nos résultats montrent l’importance de CLUH dans le maintien de l’homéostasie métabolique cellulaire, et une régulation potentielle de ses fonctions par oligomérisation. / Mitochondrial dynamics and distribution are critical insupplying ATP in response to energy demands. CLUHis a highly conserved protein involved in mitochondrial distribution, whose dysfunction leads to mitochondrial clustering around the nucleus. To gain insight into the role of CLUH in cellular metabolism, we generated CLUH knockout cells using CRISPR/Cas9. We show that mitochondrial clustering is associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in OXPHOS defects. This energetic impairment was found to be due to the alteration of mitochondrial translation, leading to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectrometry disclosed a dysfunctional Krebs cycle and an alteration of fatty acidoxidation. Thus, we established a clear function of CLUH in coupling mitochondrial distribution to the control of cellular energetic and metabolic status. To further analyze CLUH function, we performed in silico the prediction of the functional domains of this protein, disclosing 5 evolutionary conserved domains within the CLUH primary sequence. We reveal an oligomerization of CLUH into tetramers and octamers, and show a dominant negative effect associated to the expression of CLUH truncated forms missing Clu-N or TPR domains. Taken together, our studies reveal the importance of CLUH in maintaining cellular metabolism homeostasis and the potential regulation of its function through oligomerization.

O ácido indol-3-acético (AIA) é um produto do metabolismo do triptofano encontrado nos organismos animais, vegetais e em microrganismos. Destacam-se os trabalhos que atribuíram ao AIA efeitos tanto antioxidantes quanto proxidantes em diferentes sistemas biológicos. O objetivo do presente estudo foi o de avaliar os efeitos da administração do AIA no metabolismo muscular e cerebral e na atividade elétrica cerebral de ratos. Foram realizados dois grupos de experimentos. No primeiro grupo foram avaliados os seguintes parâmetros: taxa glicêmica e o ganho de peso corporal de animais tratados por 14 dias com AIA (40 mg/Kg de peso vivo, via intragástrica); atividade das enzimas antioxidantes glutationa redutase (GR), catalase (CAT) e superóxido dismutase (SOD) e das enzimas do metabolismo da glicose hexoquinase (HQ), lactato desidrogenase (LDH) e glicose-6-fosfato desidrogenase (G6PDH) nos músculos sóleo e gastrocnêmio e a atividade da enzimas antioxidantes GR, CAT e SOD e a quantificação dos produtos resultantes da peroxidação lipídica (TBARs) no cérebro de ratos tratados por 14 dias com diferentes doses de AIA (1, 18 e 40 mg/Kg de peso animal, via intragástrica). Os respectivos controles de todas essas análises foram obtidos de ratos que receberam 1 mL de tampão fosfato pH 7,4 via intragástrica sob as mesmas condições experimentais. No segundo grupo de experimentos foi obtido o eletroencefalograma (EEG) dos animais. O EEG obtido foi filtrado nas bandas de freqüências delta (0,3-4 Hz), teta (4-8 Hz), alfa (8-12 Hz) e beta (12-30 Hz) e em cada banda calculou-se a energia do sinal. Foram avaliados o EEG de animais tratados com AIA (40 mg/Kg de peso vivo) e tratados com triptofano (40 mg/Kg de peso animal), ambos por via intragástrica. Os controles para esses tratamentos foram o EEG coletado 1 hora antes e 1 hora depois da administração de 1mL de tampão fosfato por via intragástrica no mesmo animal que recebeu o tratamento. Os resultados foram analisados por ANOVA com significância de 0,05 usando o teste de Tuckey e os estimadores foram validados usando-se bootstrap. A adminitração de AIA (40mg/Kg de peso vivo) não alterou a taxa glicêmica e evolução de peso corporal dos animais, em relação ao controle. Não foram observadas diferenças significativas entre os resultados obtidos de amostras de animais tratados com AIA (todas as doses) em relação aos respectivos controles para: atividade das enzimas antioxidantes muscular e cerebral; enzimas envolvidas com o metabolismo da glicose muscular; conteúdo de peroxidação lipídica (TBARs) cerebral. O método não invasivo de aquisição de EEG desenvolvido para ratos permitiu adquirir e analisar o sinal elétrico cerebral. Não foram observadas alterações no padrão do EEG após a administração de tampão fosfato. No entanto, o AIA na dose de 40 mg/Kg de peso vivo alterou o padrão do EEG do animal, pois, a energia das freqüências de ondas alfa (8-12 Hz) e beta (12-30 Hz) foi maior em relação ao estado normal e após administração de tampão fosfato. Já o triptofano na dose de 40 mg/Kg de peso vivo aumentou a energia da onda delta (0,3-4 Hz) e diminuiu na energia da onda beta (12-30 Hz) em relação ao estado normal. O método não invasivo de EEG para rato desenvolvido neste trabalho foi sensível para detectar a atividade elétrica encefálica dos animais e o triptofano serviu como parâmetro de referência, pois promoveu diferentes alterações no padrão do EEG daquelas observadas nos animais tratados com AIA. Conclui-se que o AIA não interferiu nos parâmetros metabólicos oxidativos e energéticos dos músculos e do cérebro dos animais estudados, mas promoveu alterações fisiológicas que desencadearam as mudanças observadas na energia do sinal de EEG dos animais. / Indole-3-acetic acid (IAA) is a tryptophan metabolic found in animals organisms, microorganisms and vegetables. It is remarkable the work done to IAA antioxidants and proxidants effects in several biological systems. The main purpose of these studies was to evaluate the effects of intragastric IAA administration in brain and in muscle metabolism and electrical brain activities in rat. The experiments were done in two groups. The first one, were evaluated the following parameters: glycemic rate and corporal gain weight to those animals treated14 days with IAA (40 mg/Kg of body weight); activity of antioxidants enzymes as glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD); activities of hexokinase (HQ), lactate dehidrogenase (LDH) and glucose-6-phosphate dehidrogenase (G6PDH) on soleus and gastrocnemic muscle; antioxidants enzymes activities and level of tiobarbituric reactives subtances (TBARs) in brain from rats treated during 14 days with doses of IAA (1,18 and 40 Kg/kg body of weight). All those analyses controls were obtained from rat that was given 1 mL of phosphate buffered saline, pH 7 (PBS), under the same experiments conditions as the group treated with IAA. On the second group of experiments was evaluated EEG pattern obtained from fixed electrodes on the animal skin surface were not sedated, and shown at delta frequency (0.3-4 Hz), theta (4-8 Hz), alpha (8-12 Hz) and beta (12-30 Hz) and the energy of those band frequency was calculated using a developed algorithm software MATLAB®. EEG was evaluated from animals treated with IAA (40 mg/Kg body weight) and treated with tryptophan (40 mg/Kg body weight), both intragastric. The management control for those treatments were EEG collected 1 hour before and 1 hour after the intragstric administration of 1mL PBS at the same animal that received the treatment. The results were analysed by ANOVA with great significance of 0.05 using the Tukey test and were evaluated by bootstrap. The IAA administration (40 mg/Kg body weight) did not change the glycaemia rate and the animal weight evolution, to compare with the control. Were not observed any significant differences among results from animals treated with IAA (all doses) relating to respective controls to: a) brain and muscles antioxidants enzymes activity; b) activities of enzymes with muscular glucose metabolism; c) brain lipid peroxidation contents by TBARs level. No invasive EEG colleting methods developed for rat allowed to collect and analyse electric brain signal. After an administration of PBS, were not observed any changes at EEG pattern. IAA dose of 40 mg/Kg body weight did change the animal EEG standard, the frequency energy of alpha wave to (8-12 Hz) and beta (12-30 Hz) was higher then normal after administration of PBS. On the other side, tryptophan dose of 40 mg/Kg body weight increased the delta wave energy to (0,3-4 Hz) and decreased the beta wave energy to (12-30 Hz), to compare withfthe normal standard. Non invasion EEG colleting methods for rat developed in this studies was sensible in order to detect an animals electric encephalic activity and the tryptophan became as reference parameter, due to several changes on pattern EEG to those animals treated with IAA. Concluding that, IAA did not interfere on oxidative metabolic parameters, neither to the brain and muscles of the studied animals, but promoted physiological changes that was possible to observe on animals electroencephalogram.

Com o avanço da idade observamos a queda na eficiência do Sistema Imunológico, estando relacionada ao aumento da morbidade e mortalidade em idosos. Dentre as células do sistema imunológico encontramos os macrófagos que garantem ao organismo a capacidade de defesa contra infecções, proliferação de células tumorais e reparo de tecidos. Uma das formas de reverter ou até mesmo restaurar algumas das funções imunológicas comprometidas com o processo de envelhecimento é a utilização da prática de exercício aeróbio moderado. Por este motivo, estudamos o efeito do treinamento moderado em natação sobre a função e o metabolismo de macrófagos de ratos envelhecidos. Em macrófagos obtidos da cavidade peritoneal, observamos uma melhora da capacidade funcional, através do aumento das funções de aderência, quimiotaxia e produção de peróxido de hidrogênio (H2O2) e óxido nítrico (NO-), que foram acompanhadas pelo aumento no metabolismo de glicose (aumento de consumo e da enzima hexoquinase), contribuindo para a melhora da função imune no envelhecimento. / Disorders of the immune function contribute to the high incidence of infections and cancer among elderly people. Macrophages play a crucial role in immune response, destroying bacteria, parasites, viruses and tumour cells through various mechanisms of action. Exercise is able to induce changes and modulate the immune response. The aim of the present work was to evaluate the function and metabolism of macrophages obtained from old rats submitted to moderate exercise training. Sedentary adult (2 – 4 months), old (15 – 18 months) and trained old rats were studied. The results show an increase in the function of macrophages obtained from the peritoneal cavity of trained old rats compared with old rats, regarding chemotaxis, hydrogen peroxide and nitric oxide production, as well a enhanced glucose consumption and increased maximal activity of the enzymes hexoquinase and glutaminase. In summary, our results indicate that exercise (moderate training) stimulates some functional aspects of macrophages of old rats, with a concomitant increase in glucose metabolism.

A utilização de células humanas para a produção do fator VIII de coagulação recombinante (rFVIII) visa obter padrões de glicosilação equivalentes aos encontrados na proteína normal. O objetivo do trabalho foi obter um processo de produção do rFVIII em biorreator em perfusão, devido à sua labilidade térmica. Foram realizados estudos preliminares em Spinner e biorreator utilizando uma linhagem de rHeLa, cujos resultados embasaram os estudos com a linhagem produtora rSkHep. Foram utilizados microcarregadores nos cultivos com esta linhagem devido à dificuldade de adaptação da mesma à suspensão. Ensaios preliminares identificaram a melhor condição de cultivo com 3 g/L Mic e 1 cel/mic e, a partir destes valores, realizou-se um ensaio em perfusão, com tempo de residência de 24 h, no qual as variáveis controladas foram mantidas constantes durante três tempos de residência. A concentração de rFVIII obtida foi semelhante 2 UI/ mL. / The interest in using human cells for the recombinant coagulation factor VIII (rFVIII) lies in obtaining glycosylation patterns similar to the ones found in the normal protein. The objective of this work was to obtain a process for rFVIII production in bioreactor, in perfusion mode, due to the thermal lability of the protein. Using a recombinant HeLa cell line adapted to suspension growth a group of studies in a bioreactor in batch mode were performed. These results were the basis for the studies performed with the producing cell line rSkHep. Microcarriers (micc) were used due to the harshness to adapt the cell line to suspension and to serum-free medium. Preliminary tests identified the best culture condition with 3 g micc/L and 3 cell/micc and, from its values, it was performed a bioreactor study in perfusion mode, with a residence time of 24 hours. The controlled variables were kept constant for three residence times. The maximum rFVIII concentration obtained was 2 UI/mL.

While metabolic enzymes inside the cell nucleus were initially considered “contaminants”, recent evidence has shown that these fulfill essential functions in epigenetic regulation. Indeed a model is emerging in which local metabolite pools influence various nuclear processes. In this model, the subcellular distribution and organization of metabolic factors have a crucial role in the complex logic and regulation of nuclear functions. Cancer cells exploit nuclear metabolic enzymes to alter the synthesis and utilization of metabolites that sustain their transcriptional programs allowing their abnormal proliferation. Understanding the precise molecular mechanisms that modulate the distribution of nuclear metabolic enzymes and their related biological functions has the potential to uncover novel therapeutic vulnerabilities of malignant cells. Here, we describe an unexpected subcellular distribution of acetyl-CoA carboxylase alpha (ACC1), the rate-limiting enzyme of de novo fatty acid synthesis. We found that in cancer cells, ACC1 is not restricted to the cytoplasm. Instead, at mitosis and after the nuclear envelope breakdown, it transiently redistributes into filament-like structures that contact condensed chromosomes. Simultaneous profiling of protein-protein and -DNA interactions defined ACC1 association with different factors associated with the cellular machinery that modulates chromosome segregation, including the centromere, the kinetochore, and the fibrous corona. Inducible depletion of ACC1 resulted in altered mitotic progression and accumulation of chromosome segregation defects – effects that are abolished only with the reconstituted expression of catalytically active mutants of ACC1 but not its inactive counterparts. We further found that the abundance of malonyl-CoA – the main product of ACC1 enzymatic activity – gradually increases towards the onset of mitosis, being a significant determinant for histone malonylation. Overall we uncovered a previously unknown function of ACC1 in modulating mitotic progression and chromosome segregation. Our findings support a model where local niches of malonyl-CoA might act as signal molecules for faithful chromosome segregation.

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<div>L-phenylalanine (Phe) is an important amino acid which is the precursor of various plant secondary metabolisms. Its biosynthesis and consumption are governed by different levels of regulatory mechanisms, yet our understanding to them are still far from complete. The plant has evolved a complex regulation over Phe, likely due to the fact that a significant portion of carbon assimilated by photosynthesis is diverted to its downstream products. In particular, lignin as one of them, is among the most abundant polymers in plant secondary cell wall. Studies have unraveled the interconnected metabolism involved in lignin biosynthesis, and a hierarchical gene regulatory network on top of it is also being uncovered by different research groups. These biological processes function together for sufficient lignification to ensure cell wall hydrophobicity and rigidity for plant normal growth. Yet on the other hand, the presence of lignin hinders the efficient saccharification process for biofuel production. Therefore, it is fundamental to understand lignin biosynthesis and its upstream Phe biosynthesis in a systematic way, to guide rational metabolic engineering to either reduce lignin content or manipulate its composition <i>in planta</i>.</div><div> </div><div> Phe biosynthesis was predominantly existed in plastids according to previous studies, and there exists a cytosolic synthetic route as well. Yet how two pathways are metabolically coordinated are largely under-explored. Here I describe a flux analysis using time course datasets from ¹⁵N L-tyrosine (Tyr) isotopic labeling studies to show the contributions from two alternative Phe biosynthetic routes in Petunia flower. The flux split between cytosolic and plastidial routes were sensitive to genetic perturbations to either upstream chorismate mutase within shikimate pathway, or downstream plastidial cationic amino-acid transporter. These results indicate the biological significance of having an alternative biosynthetic route to this important amino acid, so that defects of the plastidial route can be partially compensated to maintain Phe homeostasis.</div><div> </div><div> To understand the metabolic dynamics of the upstream part of lignin biosynthesis, we developed a multicompartmental kinetic model of the general phenylpropanoid metabolism in Arabidopsis basal lignifying stems. The model was parameterized by Markov Chain Monte Carlo sampling, with data from feeding plants with ring labeled [¹³C₆]-Phe. The existence of vacuole storage for both Phe and <i>p</i>-coumarate was supported by an information theoretic approach. Metabolic control analysis with the model suggested the plastidial cationic amino-acid transporter to be the step with the highest flux controlling coefficient for lignin deposition rate. This model provides a deeper understanding of the metabolic connections between Phe biosynthesis and phenylpropanoid metabolism, suggesting the transporter step to be the promising target if one aims to manipulate lignin pathway flux.</div><div> </div><div> Hundreds of gene regulatory interactions between transcription factors and structural genes involved in lignin biosynthesis has been reported with different experimental evidence in model plant Arabidopsis, however, a public database is missing to summarize and present all these findings. In this work, we documented all reported gene regulatory interactions in Arabidopsis lignin biosynthesis, and ended up with a gene regulatory network consisting of 438 interactions between 72 genes. A network is then constructed with linear differential equations, and its parameters were estimated and evaluated with RNA-seq datasets from 13 genetic backgrounds in Arabidopsis basal stems. We combined this network with a kinetic model of lignin biosynthesis starting from Phe and ending with all monolignols participated in lignin polymerization. This hierarchical kinetic model is the first model integrating dynamic information between transcriptional machinery and metabolic network for lignin biosynthesis. We showed that it is able to provide mechanistic explanations for most of experimental findings from different genotypes. It also provides the opportunity to systematically test all possible genetic manipulation strategies targeting to lignification relevant genes to predict the lignin phenotypes <i>in silico</i>.</div>