Academic literature on the topic 'Metabolismo bioenergetico'

PurposeIntrinsic laryngeal skeletal muscle bioenergetics, the means by which muscles produce fuel for muscle metabolism, is an understudied aspect of laryngeal physiology with direct implications for voice habilitation and rehabilitation. The purpose of this review is to describe bioenergetic pathways identified in limb skeletal muscle and introduce bioenergetic physiology as a necessary parameter for theoretical models of laryngeal skeletal muscle function.MethodA comprehensive review of the human intrinsic laryngeal skeletal muscle physiology literature was conducted. Findings regarding intrinsic laryngeal muscle fiber complement and muscle metabolism in human models are summarized and exercise physiology methodology is applied to identify probable bioenergetic pathways used for voice function.ResultsIntrinsic laryngeal skeletal muscle fibers described in human models support the fast, high-intensity physiological requirements of these muscles for biological functions of airway protection. Inclusion of muscle bioenergetic constructs in theoretical modeling of voice training, detraining, fatigue, and voice loading have been limited.ConclusionsMuscle bioenergetics, a key component for muscle training, detraining, and fatigue models in exercise science, is a little-considered aspect of intrinsic laryngeal skeletal muscle physiology. Partnered with knowledge of occupation-specific voice requirements, application of bioenergetics may inform novel considerations for voice habilitation and rehabilitation.

Deficient bioenergetic signaling contributes to myocardial dysfunction and electrical instability in both atrial and ventricular cardiac chambers. Yet, approaches capable to prevent metabolic distress are only partially established. Here, in a canine model of tachycardia-induced congestive heart failure, we compared atrial and ventricular bioenergetics and tested the efficacy of metabolic rescue with the vasopeptidase inhibitor omapatrilat. Despite intrinsic differences in energy metabolism, failing atria and ventricles demonstrated profound bioenergetic deficiency with reduced ATP and creatine phosphate levels and compromised adenylate kinase and creatine kinase catalysis. Depressed phosphotransfer enzyme activities correlated with reduced tissue ATP levels, whereas creatine phosphate inversely related with atrial and ventricular load. Chronic treatment with omapatrilat maintained myocardial ATP, the high-energy currency, and protected adenylate and creatine kinase phosphotransfer capacity. Omapatrilat-induced bioenergetic protection was associated with maintained atrial and ventricular structural integrity, albeit without full recovery of the creatine phosphate pool. Thus therapy with omapatrilat demonstrates the benefit in protecting phosphotransfer enzyme activities and in preventing impairment of atrial and ventricular bioenergetics in heart failure.

The brain is the most energy-consuming organ of the body and impairments in brain energy metabolism will affect neuronal functionality and viability. Brain aging is marked by defects in energetic metabolism. Abnormal tau protein is a hallmark of tauopathies, including Alzheimer’s disease (AD). Pathological tau was shown to induce bioenergetic impairments by affecting mitochondrial function. Although it is now clear that mutations in the tau-coding gene lead to tau pathology, the causes of abnormal tau phosphorylation and aggregation in non-familial tauopathies, such as sporadic AD, remain elusive. Strikingly, both tau pathology and brain hypometabolism correlate with cognitive impairments in AD. The aim of this review is to discuss the link between age-related decrease in brain metabolism and tau pathology. In particular, the following points will be discussed: (i) the common bioenergetic features observed during brain aging and tauopathies; (ii) how age-related bioenergetic defects affect tau pathology; (iii) the influence of lifestyle factors known to modulate brain bioenergetics on tau pathology. The findings compiled here suggest that age-related bioenergetic defects may trigger abnormal tau phosphorylation/aggregation and cognitive impairments after passing a pathological threshold. Understanding the effects of aging on brain metabolism may therefore help to identify disease-modifying strategies against tau-induced neurodegeneration.

Blood-based bioenergetic profiling provides a minimally invasive assessment of mitochondrial health shown to be related to key features of aging. Previous studies show that blood cells recapitulate mitochondrial alterations in the central nervous system under pathological conditions, including the development of Alzheimer’s disease. In this study of nonhuman primates, we focus on mitochondrial function and bioenergetic capacity assessed by the respirometric profiling of monocytes, platelets, and frontal cortex mitochondria. Our data indicate that differences in the maximal respiratory capacity of brain mitochondria are reflected by CD14+ monocyte maximal respiratory capacity and platelet and monocyte bioenergetic health index. A subset of nonhuman primates also underwent [18F] fluorodeoxyglucose positron emission tomography (FDG-PET) imaging to assess brain glucose metabolism. Our results indicate that platelet respiratory capacity positively correlates to measures of glucose metabolism in multiple brain regions. Altogether, the results of this study provide early evidence that blood-based bioenergetic profiling is related to brain mitochondrial metabolism. While these measures cannot substitute for direct measures of brain metabolism, provided by measures such as FDG-PET, they may have utility as a metabolic biomarker and screening tool to identify individuals exhibiting systemic bioenergetic decline who may therefore be at risk for the development of neurodegenerative diseases.

The rising incidence of metabolic diseases worldwide has prompted renewed interest in the study of intermediary metabolism and cellular bioenergetics. The application of modern biochemical methods for quantitating fuel substrate metabolism with advanced mouse genetic approaches has greatly increased understanding of the mechanisms that integrate energy metabolism in the whole organism. Examination of the intermediary metabolism of skeletal cells has been sparked by a series of unanticipated observations in genetically modified mice that suggest the existence of novel endocrine pathways through which bone cells communicate their energy status to other centers of metabolic control. The recognition of this expanded role of the skeleton has in turn led to new lines of inquiry directed at defining the fuel requirements and bioenergetic properties of bone cells. This article provides a comprehensive review of historical and contemporary studies on the metabolic properties of bone cells and the mechanisms that control energy substrate utilization and bioenergetics. Special attention is devoted to identifying gaps in our current understanding of this new area of skeletal biology that will require additional research to better define the physiological significance of skeletal cell bioenergetics in human health and disease.

Mitochondrial bioenergetic function is a central component of cellular metabolism in health and disease. Mitochondrial oxidative phosphorylation is critical for maintaining energetic homeostasis, and impairment of mitochondrial function underlies the development and progression of metabolic diseases and aging. However, measurement of mitochondrial bioenergetic function can be challenging in human samples due to limitations in the size of the collected sample. Furthermore, the collection of samples from human cohorts is often spread over multiple days and locations, which makes immediate sample processing and bioenergetics analysis challenging. Therefore, sample selection and choice of tests should be carefully considered. Basic research, clinical trials, and mitochondrial disease diagnosis rely primarily on skeletal muscle samples. However, obtaining skeletal muscle biopsies requires an appropriate clinical setting and specialized personnel, making skeletal muscle a less suitable tissue for certain research studies. Circulating white blood cells and platelets offer a promising primary tissue alternative to biopsies for the study of mitochondrial bioenergetics. Recent advances in frozen respirometry protocols combined with the utilization of minimally invasive and non-invasive samples may provide promise for future mitochondrial research studies in humans. Here we review the human samples commonly used for the measurement of mitochondrial bioenergetics with a focus on the advantages and limitations of each sample.

Alterations in cardiac energy metabolism play a key role in the pathogenesis of diabetic cardiomyopathy. Hypercholesterolemia associated with bioenergetic impairment and oxidative stress has not been well characterized in the cardiac function under glycemic control deficiency conditions. This work aimed to determine the cardioprotective effects of quercetin (QUE) against the damage induced by a high-cholesterol (HC) diet in hyperglycemic rats, addressing intracellular antioxidant mechanisms and bioenergetics. Quercetin reduced HC-induced alterations in the lipid profile and glycemia in rats. In addition, QUE attenuated cardiac diastolic dysfunction (increased E:A ratio), prevented cardiac cholesterol accumulation, and reduced the increase in HC-induced myocyte density. Moreover, QUE reduced HC-induced oxidative stress by preventing the decrease in GSH/GSSG ratio, Nrf2 nuclear translocation, HO-1 expression, and antioxidant enzymatic activity. Quercetin also counteracted HC-induced bioenergetic impairment, preventing a reduction in ATP levels and alterations in PGC-1α, UCP2, and PPARγ expression. In conclusion, the mechanisms that support the cardioprotective effect of QUE in rats with HC might be mediated by the upregulation of antioxidant mechanisms and improved bioenergetics on the heart. Targeting bioenergetics with QUE can be used as a pharmacological approach to modulate structural and functional changes of the heart under hypercholesterolemic and hyperglycemic conditions.

Circulating immune cells are considered a source for biomarkers in health and disease, since they are exposed to nutritional, metabolic and immunological stimuli in the vasculature. Cryopreservation of leucocytes is routinely used for long-term storage and determination of phenotypic/functional changes at a later date. Exploring the role of bioenergetics and mitochondrial (dys)function in leucocytes is often examined by using freshly isolated cells. The aim of the pilot study described herein was to assess leucocyte bioenergetics in cryopreserved cells. Leucocytes were isolated from whole blood, counted and frozen in liquid nitrogen (LN2) for a period of 3 months. Cells were thawed at regular intervals and bioenergetic analysis performed using the Seahorse XFe96 flux analyser. Cryogenic storage reduced cell viability by 20%, but cell bioenergetic responses were largely intact for up to 1 month storage in LN2. However, after 1 month storage, mitochondrial function was impaired as reflected by decreasing basal respiration, ATP production, maximum (MAX) respiration, reserve capacity and coupling efficiency. Conversely, glycolytic activity was increased after 1 month, most notably the enhanced glycolytic response to 25 mM glucose without any change in glycolytic capacity. Finally, calculation of bioenergetic health index (BHI) demonstrated that this potential diagnostic parameter was sensitive to cryopreservation. The present study has demonstrated for the first time that cryopreservation of primary immune cells modified their metabolism in a time-dependent fashion, indicated by attenuated aerobic respiration and enhanced glycolytic activity. Taken together, we recommend caution in the interpretation of bioenergetic responses or BHI in cryopreserved samples.

AbstractIt is now becoming clear that human metabolism is extremely plastic and varies substantially between healthy individuals. Understanding the biochemistry that underlies this physiology will enable personalized clinical interventions related to metabolism. Mitochondrial quality control and the detailed mechanisms of mitochondrial energy generation are central to understanding susceptibility to pathologies associated with aging including cancer, cardiac and neurodegenerative diseases. A precision medicine approach is also needed to evaluate the impact of exercise or caloric restriction on health. In this review, we discuss how technical advances in assessing mitochondrial genetics, cellular bioenergetics and metabolomics offer new insights into developing metabolism-based clinical tests and metabolotherapies. We discuss informatics approaches, which can define the bioenergetic-metabolite interactome and how this can help define healthy energetics. We propose that a personalized medicine approach that integrates metabolism and bioenergetics with physiologic parameters is central for understanding the pathophysiology of diseases with a metabolic etiology. New approaches that measure energetics and metabolomics from cells isolated from human blood or tissues can be of diagnostic and prognostic value to precision medicine. This is particularly significant with the development of new metabolotherapies, such as mitochondrial transplantation, which could help treat complex metabolic diseases.

Molte cellule tumorali, al fine di generare ATP e sostenere i processi anabolici, si servono principalmente della glicolisi piuttosto che della respirazione mitocondriale. Di conseguenza, il glucosio assume un ruolo critico per la sopravvivenza e la proliferazione delle cellule tumorali. Inoltre, attraverso la via dei pentosi fosfati, il glucosio porta alla formazione di NADPH, contribuendo al mantenimento nelle cellule dell’equilibrio ossidativo. Nondimeno, il glucosio può entrare anche nel pathway biosintetico delle esosammine (HBP), sostenendo la N- e O-glicosilazione di lipidi e proteine, importante per lo sviluppo tumorale. Considerando l’essenziale ruolo del glucosio, un possibile approccio per la terapia antitumorale è l’utilizzo del metabolismo del glucosio come target, non solo attraverso la glicolisi ma sfruttando anche gli altri processi glucosio-dipendenti. A tal proposito, la deprivazione di glucosio e la seguente analisi del destino cellulare a livello fenotipico e molecolare possono costituire una strategia utile per smascherare tutti i meccanismi mediati dal glucosio che partecipano alla crescita e alla sopravvivenza delle cellule tumorali. Tale strategia potrebbe essere poi sfruttata per offrire nuovi target e progettare nuove terapie antitumorali. Sebbene alcuni dati indichino che i tumori originino da cellule con persistenti difetti alla catena respiratoria mitocondriale, l’inibizione della fosforilazione ossidativa (OXPHOS) sembra una condizione di adattamento più che una causa della riprogrammazione metabolica delle cellule tumorali. In questo scenario, i meccanismi di regolazione post-traduzionali, di natura essenzialmente reversibile, a carico di proteine mitocondriali potrebbero assumere un importante ruolo regolatorio. Una delle principali modificazioni post-traduzionali è la fosforilazione dei residui Ser/Thr e, a tal proposito, la chinasi PKA presenta numerosi target a livello mitocondriale ed è coinvolta nella regolazione di biogenesi, trasporto e attività dei Complessi I e IV e della morfologia mitocondriale. Poiché è stato osservato che K-ras può causare la diminuzione dell’espressione di geni codificanti per componenti della via cAMP/PKA, nelle cellule K-ras-trasformate la deregolazione di tale via potrebbe portare alla disfunzione mitocondriale ed allo switch metabolico caratteristico delle cellule tumorali. A conferma di questa ipotesi, le cellule K-ras-trasformate mostrano minori livelli di attività dell’enzima PKA rispetto alle cellule normali. Inoltre, la stimolazione esogena della attività della PKA, ottenuta mediante trattamento con forskolina (FSK), protegge le cellule K-ras-trasformate, sia murine sia umane, dalla morte indotta dalla deplezione di glucosio. Tale protezione è dovuta alla stimolazione dell’attività del Complesso I, all’aumento dell’ATP intracellulare e della fusione mitocondriale e alla riduzione dei livelli di ROS. L’inibizione specifica di PKA previene quasi completamente molti di questi effetti. Inoltre, il breve trattamento con Mdivi-1, molecola in grado di favorire la fusione mitocondriale, riduce fortemente i livelli di ROS specialmente nelle cellule trasformate, indicando una stretta relazione tra morfologia e attività mitocondriale. Queste osservazioni supportano l’idea che l’apoptosi indotta dalla deprivazione di glucosio nelle cellule K-ras-trasformate è associata alla deregolazione della via cAMP/PKA che a sua volta causa la diminuzione dell’attività del Complesso I, la riduzione della produzione di ATP e la prevalenza della fissione mitocondriale rispetto alla fusione. Tale scoperta può aprire nuovi scenari per lo sviluppo di farmaci antitumorali. Poiché la carenza di glucosio si può riscontrare nell’ambiente in cui cresce e si sviluppa il tumore, tale condizione può essere sfruttata per potenziare l’azione di specifici agenti, come alcuni modulatori dell’OXPHOS. Infatti, l’inibizione delle funzioni mitocondriali in condizioni di deprivazione di glucosio potrebbe risultare letale per le cellule tumorali. In accordo, in questo lavoro viene mostrato che la deprivazione di glucosio e gli inibitori del Complesso I, come rotenone, piericidina A e capsaicina, hanno un effetto sinergico nell’indurre la morte delle cellule tumorali. Nello specifico, basse dosi d’inibitori del Complesso I, inefficaci sulle cellule normali e su cellule cresciute in alto glucosio, diventano citotossiche per le cellule tumorali cresciute in basso glucosio. L’effetto citotossico degli inibitori del Complesso I sulle cellule tumorali è ulteriormente e fortemente aumentato quando l’attività OXPHOS viene stimolata tramite il trattamento con FSK. Queste osservazioni dimostrano che la riattivazione della funzione mitocondriale associata alla deplezione di glucosio e al trattamento con basse dosi di inibitori del Complesso I riduce fortemente la sopravvivenza delle cellule tumorali e potrebbe quindi essere valutato come approccio terapeutico. Come indicato in precedenza, nelle cellule tumorali il glucosio è implicato in numerosi processi. L’analisi trascrittomica e proteomica di cellule murine K-ras-trasformate e della loro controparte normale mostra che la deprivazione di glucosio modula l’espressione di molti geni legati allo stress del reticolo endoplasmatico e all’Unfolded Protein Response (UPR). L’attivazione di tale risposta si osserva in entrambe le linee cellulari ma più fortemente nelle cellule trasformate, dove è associata anche alla morte cellulare. Infatti, la sua attenuazione tramite l’inibitore della traduzione proteica, cicloesimide, o lo chaperone chimico, 4-fenil-butirrato, protegge specificatamente le cellule trasformate dalla morte cellulare in basso glucosio. Anche l’inibizione della chinasi proapoptotica JNK, attivata a valle dell’UPR, previene specificatamente la morte delle cellule trasformate. Questa osservazione è in accordo col fatto che in basso glucosio le cellule trasformate mostrano una maggiore attivazione di JNK rispetto alle cellule normali. Inoltre, l’attivazione dell’UPR e la morte glucosio-dipendente delle cellule trasformate è completamente prevenuta dall’aggiunta nel terreno di coltura di un substrato dell’HBP, N-Acetyl-D-glucosammina, cosa che suggerisce una stretta relazione tra i due processi. È interessante notare che anche cellule umane esprimenti l’oncogene K-ras e caratterizzate da un fenotipo iperglicolitico mostrano simili effetti in seguito alla modulazione dell’UPR o dell’HBP. Quindi, la deprivazione di glucosio nelle cellule K-ras-trasformate può indurre un meccanismo di morte cellulare UPR-dipendente, attivato dall’eccessivo accumulo di proteine mal foldate, probabilmente come conseguenza della riduzione della N-glicosilazione delle proteine. La piena delucidazione di questa risposta potrebbe essere importante per progettare nuove strategie terapeutiche antitumorali. Oggi la nuova sfida della ricerca e della terapia antitumorale è il totale sradicamento del tumore, uccidendo anche le cellule staminali tumorali (cancer stem cells, CSCs). Considerando l’importante ruolo del metabolismo e della sua riprogrammazione nello sviluppo tumorale, la caratterizzazione del metabolismo delle CSCs può essere considerata un importante mezzo per lo sviluppo di nuove strategie antitumorali. Recentemente, è stata ottenuta la linea cellulare staminale di osteosarcoma umano, 3AB-OS. In questo lavoro di tesi ho svolto una prima caratterizzazione del suo profilo metabolico, paragonato a quello delle cellule tumorali MG63, da cui le cellule 3AB-OS sono state selezionate. Si è osservato che le cellule 3AB-OS dipendono più fortemente dalla glicolisi rispetto alle cellule MG63. Infatti, quando cresciute in presenza di galattosio e piruvato (substrati mitocondriali) le cellule 3AB-OS riducono maggiormente la propria capacità proliferativa rispetto alle cellule MG63. Esse risultano anche essere fortemente sensibili alla deprivazione di glucosio e al trattamento con inibitori della glicolisi mentre sono insensibili all’inibizione della catena respiratoria. Inoltre, diversamente dalle cellule MG63, le cellule 3AB-OS presentano principalmente mitocondri frammentati, in particolare in basso glucosio. Tutte queste osservazioni suggeriscono che il metabolismo energetico delle cellule 3AB-OS presenti caratteristiche paragonabili a quello delle cellule staminali normali e delle cellule tumorali caratterizzate da un metabolismo glicolitico. Può essere interessante notare che il profilo trascrizionale delle cellule 3AB-OS è simile a quello delle cellule K-ras-trasformate, confermando la similitudine tra le CSCs e le cellule tumorali glicolitiche. Quindi, alcune strategie sviluppate per il trattamento delle cellule tumorali glucosio-dipendenti potrebbero essere usate anche per trattare specifiche CSCs.
Several cancer cells, in order to generate ATP and sustain different anabolic processes, rely mainly on glycolysis instead of Oxidative Phosphorylation (OXPHOS). Thus, glucose assumes a critical role for cancer cell survival and proliferation. Moreover, through the penthose phospate pathway glucose leads to production of NADPH contributing to maintenance of cellular oxidative equilibrium. Besides, glucose can also enter Hexosamine Biosynthesis Pathway (HBP), sustaining lipid and protein N- and O-glycosylation that cover an important role in cancer development. Taking in consideration the essential role of glucose in cancer, one important anticancer therapeutic approach is to target its metabolism namely glycolysis and the other processes in which it is involved. On this regard, glucose deprivation and consequent analysis of cancer cell fate both at phenotypical and molecular level can be a useful strategy to unmask all mechanisms that participate to glucose-mediated cancer cell growth and survival. Such a strategy could be subsequently exploited to provide new targets and to set new anticancer therapies. Although there is evidence that tumors originate from cells with persistent defects in the mitochondrial respiratory system, inhibition of OXPHOS activity seems to be an adaptation to cancer metabolism reprogramming rather than a cause. In this scenario, reversible post-translational modifications of mitochondrial components could assume an important regulatory role. Among the most important post-translational modifications there is Ser/Thr phosphorylation and, on this regard, the protein kinase PKA has numerous mitochondrial targets being involved in the regulation of the biogenesis, the import and the activity of mitochondrial Complex I or IV as well as of mitochondrial morphology. Since it has been observed that oncogenic K-ras may lead to a depression of genes encoding for components of the cAMP/PKA signaling pathway, in K-ras-transformed cells the deregulation of cAMP/PKA pathway could cause OXPHOS depression and “glucose addiction” of cancer cells. In agreement with such a hypothesis, K-ras-transformed cells show lower PKA activity as compared to normal cells. Moreover, exogenous stimulation of PKA activity, achieved by Forskolin (FSK) treatment, protects mouse and human K-ras-transformed cells from apoptosis induced by glucose deprivation, by enhancing Complex I activity, intracellular ATP levels and mitochondrial fusion and by decreasing intracellular ROS levels. Worth noting, several of these effects are almost completely prevented by inhibition of PKA activity. Moreover, short time treatment with Mdivi-1, a molecule that favors mitochondrial fusion, strongly decreases the cellular ROS levels especially in transformed cells, indicating a close relationship between mitochondrial morphology and activity. These findings support the notion that glucose shortage-induced apoptosis, specific of K-ras-transformed cells, is associated to a derangement of PKA signaling that leads to mitochondrial Complex I decrease, reduction of ATP formation and prevalence of mitochondrial fission over fusion. Such a discovery can thereby open new approaches for the development of anticancer drugs. Given that glucose shortage is often encountered in the tumor microenvironment, it can be exploited to potentiate the action of specific agents, such as the mitochondrial OXPHOS activity modulators, that in condition of glucose deprivation could be lethal for cancer cells. Accordingly, it is shown that glucose deprivation and Complex I inhibitors, i.e., rotenone, piericidin A and capsaicin, synergize in inducing cancer cell death. In particular, low doses of Complex I inhibitors, ineffective on normal cells and on cells grown in high glucose, become specifically cytotoxic on cancer cells cultured in low glucose. Importantly, the cytotoxic effect of Complex I inhibitors is strongly enhanced when mitochondrial OXPHOS activity is stimulated by FSK. These findings demonstrate that the reactivation of the mitochondrial function associated with glucose depletion and low doses of mitochondrial Complex I inhibitors strongly affect cancer cell survival. This therapeutic approach might be valuable to eradicate cancer cells. As above indicated, glucose is implicated in numerous processes in cancer cells. Transcriptomic and proteomic analyses applied to mouse K-ras-transformed cells as compared to normal cells show that glucose deprivation modulates the expression of several genes linked to endoplasmic reticulum stress and the Unfolded Protein Response (UPR). The activation of such a response, as confirmed by mRNA and protein expression, is observed in both cell lines, but only in transformed cells is strictly associated to their death. In fact, its attenuation by protein translation inhibitor cycloheximide or chemical chaperone 4-Phenyl-butyrate specifically rescues transformed cells from death. Moreover, glucose deprivation-induced transformed cell death is also prevented by inhibition of an UPR downstream pro-apoptotic kinase, JNK, whose activation is observed specifically in transformed cells as compared to normal cells. Interestingly, UPR activation and death of transformed cells is completely prevented by addition of a specific HBP substrate, namely N-Acetyl-D-glucosamine, suggesting a strict relation between the two processes. Notably, also oncogenic K-ras expressing human glycolytic cells show similar effects after UPR modulating treatments. Thus, we show that glucose deprivation can induce an UPR-dependent transformed cell death mechanism, which is activated by harmful accumulation of unfolded proteins, probably as consequence of N-glycosylation protein reduction. The full elucidation of this response could be relevant to design new therapeutic strategies. Today the new challenge of anticancer research and therapy is the total eradication of the cancer, targeting cancer stem cells (CSCs). Considering the important role of metabolism and metabolic reprogramming in cancer development, also the definition of CSCs metabolism can be considered an important tool for future strategies targeting these cells. Recently, a human osteosarcoma 3AB-OS CSC-like line has been developed. Therefore we have decided to characterize its metabolic features as compared to the parental osteosarcoma MG63 cells, from which 3AB-OS cells were previously selected. 3AB-OS cells depend on glycolytic metabolism more strongly than MG63 cells. Indeed, addition to the growth medium of galactose and pyruvate -mitochondrial specific substrates- instead of glucose markedly reduces 3AB-OS growth, as compared to MG63 cells. In line with these findings 3AB-OS cells, compared to MG63 cells, are strongly sensitive to glucose depletion, glycolysis inhibition and less sensitive to respiratory inhibitors. Additionally, in contrast to MG63 cells, 3AB-OS display mainly fragmented mitochondria, particularly in low glucose. Overall, these findings suggest that 3AB-OS energy metabolism is more similar either to normal stem cells or to cancer cells characterized by a glycolytic metabolism. Interestingly, the transcriptional profile of CSCs is similar to that of K-ras-transformed cells, confirming a possible similarity to glycolytic cancer cells. Therefore, some strategies developed for glucose addicted cancer cells could be used also to treat specific CSCs.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O imidacloprido (IMD) é um inseticida neonicotinóide largamente utilizado em diversas culturas agrícolas e em animais para o controle de pragas. O IMD é rapidamente absorvido pelo trato gastrointestinal e por contato, sendo rápida e uniformemente distribuído nos órgãos e tecidos. Dados da literatura mostram que as concentrações mais elevadas foram observadas nos órgãos de eliminação: fígado e rins. O fígado é o principal órgão envolvido na biotransformação de substâncias exógenas (xenobióticos), convertendo compostos hidrofóbicos em hidrossolúveis, mais facilmente eliminados pelo organismo. Vários estudos vêm sendo conduzidos sobre os efeitos tóxicos do IMD em animais, causando danos ao fígado. Nesse sentido, o objetivo desse estudo foi avaliar os mecanismos envolvidos na toxicidade do IMD sobre a bioenergética de mitocôndrias e hepatócitos isolados de rato e ações do IMD sobre o metabolismo de carboidratos e proteínas em fígado de rato em perfusão. Em mitocôndrias isoladas, o IMD promoveu uma diminuição dose-dependente no estado 3 da respiração e na produção de ATP, sem afetar o potencial de membrana mitocondrial. Experimentos subsequentes medindo o consumo de oxigênio mostraram que o IMD não afeta a cadeia respiratória e que seu efeito é semelhante ao da oligomicina (inibidora da FoF1-ATP sintase) e/ou ao do atractilosídeo (inibidor do translocador de nucleotídeos de adenina, ANT). IMD inibiu a atividade da FoF1-ATP sintase em mitocôndrias rompidas e inibiu parcialmente a despolarização do potencial de membrana induzida pelo ADP. Esses resultados indicam que o IMD afeta a bioenergética mitocondrial por meio da inibição da FoF1-ATP sintase. Em Experimentos com hepatócitos isolados de rato os resultados da respiração foram semelhantes aos encontrados nas mitocôndrias isoladas, porém o IMD afetou a produção intracelular de ATP e induziu a morte celular somente nos hepatócitos isolados de ratos previamente tratados com dexametasona, um ativador do citocromo P450. No fígado de rato em perfusão o IMD também inibiu a produção de glicose por meio da gliconeogênese. Esses resultados sugerem que a toxicidade do IMD pode estar associada a alterações no metabolismo energético celular sendo a enzima FoF1-ATP sintase o principal alvo da ação tóxica deste inseticida, e que os metabólitos formados na biotransformação do IMD podem ser mais tóxicos do que o próprio IMD.
Imidacloprid (IMD) is a neonicotinoid insecticide widely used in various crops and animals for pest control. IMD is rapidly absorbed by the gastrointestinal tract, being rapidly and evenly distributed in the organs and tissues. The highest concentrations were observed in the elimination organs: liver and kidneys. The liver is the main organ involved in the biotransformation of exogenous substances (xenobiotics), with the capacity to convert hydrophobic compounds into water soluble metabolites, which are more easily eliminated by the organism. Studies have been conducted on the toxic effects of IMD on animals, causing damage to the liver. In this sense, the objective of this study was to evaluate the mechanisms involved in the toxicity of IMD on the bioenergetics of mitochondria and isolated hepatocytes of rats and its actions on the metabolism of carbohydrates and proteins in liver of rats in perfusion. In isolated mitochondria, IMD promoted a dose-dependent decrease in the state 3 of mitochondrial respiration and ATP levels, without affecting mitochondrial membrane potential. Subsequent experiments measuring oxygen consumption have shown that IMD does not affect the electron transport chain and that its effect is similar to that of oligomycin (FoF1-ATP synthase inhibitor) and/or atracytoside (ANT adenine nucleotide translocator inhibitor). In the perfusion rat liver IMD inhibited the activity of FoF1-ATP synthase in freeze/thaw-disrupted mitochondria and partially inhibited the depolarization of the membrane potential induced by ADP. These results indicate that IMD affects in mitochondrial bioenergetics by inhibiting FoF1-ATP synthase. In experiments with isolated hepatocytes respiration results were similar to those found in isolated mitochondria, but IMD affected the intracellular production of ATP and induced cell death only in hepatocytes isolated from rats previously treated with dexamethasone, a cytochrome P450 activator. IMD also inhibited the production of glucose by gluconeogenesis. These results suggest that IMD toxicity may be associated with changes in cellular energy metabolism with the enzyme FoF1-ATP synthase being the main target of the toxic action of this insecticide, and that the metabolites formed in the biotransformation of the IMD may be more toxic than the IMD itself.
FAPESP: 2015/19549-8

Aquest és el primer informe que demostra que l'autofàgia està mecànicament vinculat al manteniment de les cèl•lules tumorals que expressen alts nivells de CD44 i baixos nivells de CD24, que són típics de les cèl•lules mare del càncer de mama. Els nostres resultats actuals proporcionen una nova visió de com la divisió mitocondrial s'integra a la xarxa de la transcripció impulsada per factors de reprogramació, especifica de la pluripotència única de les cèl•lules mare. L'autofàgia pot controlar la refractarietat de novo de carcinomes de mama amb el gen HER2 amplificat per l'anticòs monoclonal trastuzumab (Herceptin). Per tant, el tractament de combinació amb trastuzumab i cloroquina, com a fàrmac anti-malàric i inhibidor de l’autofàgia, suprimeix radicalment el creixement del tumor en un xenoempelt de tumor completament refractari a trastuzumab en un model murí. L’addició de cloroquina amb els règims amb trastuzumab pot, per tant, millorar els resultats en les dones amb càncer de mama HER2. Aquesta és una àrea molt emocionant i molt prometedora de la investigació del càncer, com la modulació farmacològica de l'autofàgia sembla augmentar l'eficàcia dels règims contra el càncer disponibles en l'actualitat i s'obre el camí per al desenvolupament de noves estratègies terapèutiques combinatòries que s'espera que contribueixin a l'eradicació del càncer.
This is the first report demonstrating that autophagy is mechanistically linked to the maintenance of tumor cells expressing high levels of CD44 and low levels of CD24, which are typical of breast cancer stem cells. Our current findings provide new insight into how mitochondrial division is integrated into the reprogramming of the factors-driven transcriptional network that specifies the unique pluripotency of stem cells. Autophagy may control the de novo refractoriness of HER2 gene-amplified breast carcinomas to the monoclonal antibody trastuzumab (Herceptin). Accordingly, treatment with trastuzumab and chloroquine, as antimalarial drug and inhibitor of autophagy, radically suppresses tumor growth in a tumor xenograft completely refractory to trastuzumab in a mouse model. Adding chloroquine to trastuzumab-based regimens may therefore improve outcomes among women with autophagy-addicted HER2-positive breast cancer. This is a very exciting and highly promising area of cancer research, as pharmacologic modulation of autophagy appears to augment the efficacy of currently available anticancer regimens and opens the way to the development of new combinatorial therapeutic strategies that will hopefully contribute to cancer eradication.

Thesis: Ph. D. in Biochemistry, Massachusetts Institute of Technology, Department of Biology, February, 2021
Cataloged from the official PDF of thesis. "February 2021." Vita. Page 179 blank.
Includes bibliographical references.
Cellular growth and proliferation necessitates the transformation of cell-external nutrients into biomass. Strategies of biomass accumulation across the kingdoms of life are diverse and range from carbon fixation by autotrophic organisms to direct biomass incorporation of consumed nutrients by heterotrophic organisms. The goal of this dissertation is to better understand the divergent and convergent modes of metabolism that support biomass accumulation and proliferation in eukaryotic cells. We first determined that the underlying mechanism behind why rapidly proliferating cells preferentially ferment the terminal glycolytic product pyruvate is due to an intrinsic deficiency of respiration to regenerate electron acceptors. We tested this model across an assorted array of proliferating cells and organisms ranging from human cancer cells to the baker's yeast Saccharomyces cerevesiae. We next determined that a major metabolic pathway of avid electron acceptor consumption in the context of biomass accumulation is the synthesis of lipids. Insights from this work has led to the realization that net-reductive pathways such as lipid synthesis may be rate-limited by oxidative reactions. Lastly, we established the green algae Chlorella vulgaris as a model system to study the comparative metabolism of photoautotrophic and heterotrophic growth. We determined that heterotrophic growth of plant cells is associated with aerobic glycolysis in a mechanism that may be suppressed by light. Collectively, these studies contribute to a more holistic understanding of the bioenergetics and metabolic pathways employed by eukaryotic cells to accumulate biomass and lay the foundation for future studies to understand proliferative metabolism.
by Zhaoqi Li.
Ph. D. in Biochemistry
Ph.D.inBiochemistry Massachusetts Institute of Technology, Department of Biology

Bilirubin is a haem catabolite that is excreted through the hepatobiliary pathway and is therefore, commonly used as a biomarker of hepatic dysfunction and haemolysis in the clinical setting [1]. Although, bilirubin has been considered toxic [2], recent evidence suggests that mildly elevated circulating bilirubin concentrations may be protective against obesity, cardiovascular diseases (CVDs) and all-cause mortality [3–5]. Generally, the protective effects of bilirubin are attributed to its antioxidant potential [6–8], however, recent studies demonstrate that bilirubin modulates lipid metabolism and reduces adiposity, which could partly contribute to CVD protection [5,9–12]. However, a shortage of studies have examined the precise mechanisms of cholesterol metabolism and adiposity that could be affected by bilirubin. The main aims of this thesis were to: 1) determine whether hyperbilirubinaemia affects cholesterol synthesis, transport, and excretion; 2) explore bilirubin’s impact on body composition and bioenergetics including mitochondrial function in liver/skeletal muscle and changes in mitochondrial density and quality; 3) determine the effectiveness of oral Legalon® ingestion on circulating bilirubin concentrations, to investigate whether inducing mild hyperbilirubinaemia could impact circulating lipid concentrations in human participants. The first study measured the effect of hyperbilirubinaemia in mutant Gunn rats on circulating lipid concentrations, cholesterol synthesis, lipid excretion, and expression of hepatic genes/proteins involved in cholesterol metabolism. Female hyperbilirubinaemic (Gunn) rats had reduced serum cholesterol concentrations (0.60 ± 0.12 vs 1.56 ± 0.34 mM, P<0.001), elevated cholesterol synthesis (33.8 ± 3.77 vs 28.4 ± 5.73 % [13C]-cholesterol, P<0.05), enhanced LDL receptor (LDLr; P<0.01) expression, and increased biliary cholesterol excretion (232 ± 32.7 vs 141 ± 42.1 nmol hr-1 100g-1 bodyweight, P<0.001) compared to female normobilirubinaemic littermate (control) rats. These results indicate that female hyperbilirubinaemic Gunn rats have reduced circulating cholesterol in association with elevated LDLr expression. Increased LDLr expression and cholesterol synthesis is typical when hepatic cholesterol concentrations are decreased [13,14]. Therefore, increased cholesterol synthesis and LDLr expression observed in female Gunn rats may represent a counter-regulatory mechanism to maintain hepatic cholesterol content in the presence of elevated biliary cholesterol excretion [13,14]. The underlying mechanism explaining increased biliary lipid excretion in female Gunn rats remains unknown. However, this observation could be partly explained by greater relative biliary lipid (cholesterol+phospholipid) to bile acid excretion (0.33 ± 0.06 vs 0.24 ± 0.03 mol:mol, lipid:bile acids, P<0.01) in female Gunn rats. Previous studies have established that organic anions including bilirubin glucuronides disrupt the capacity of bile acid micelles to excrete lipids in the bile [15]. Biliary excretion of bilirubin conjugates was decreased in female (13.1 ± 2.92 vs 33.5 ± 5.09 nmol hr-1 100g-1 bodyweight, P<0.001) and male (11.0 ± 2.43 vs 43.2 ± 12.8 nmol hr-1 100g-1 bodyweight, P<0.001) Gunn rats compared to controls, due to UGT1A1 dysfunction and the inability to conjugate bilirubin. Therefore, decreased biliary excretion of bilirubin conjugates, as observed in Gunn rats, may potentially facilitate the greater coupled excretion of biliary lipids to bile acids as demonstrated in this study. It should be noted that this conclusion does not completely explain the results reported here because Gunn rats demonstrated significant sexual dimorphism in cholesterol metabolism. Male Gunn rats exhibited a non-significant reduction in circulating cholesterol concentrations (1.41 ± 0.15 vs 1.56 ± 0.23, P=0.14) and increased biliary lipid:bile acid excretion (0.31 ± 0.07 vs 0.25 ± 0.04 mol:mol, lipid:bile acid, P=0.08) compared to male normobilirubinaemic littermate (control) rats, indicating that additional mechanisms, beyond bilirubin excretion, are involved. For example, UGT1A1, which conjugates bilirubin also conjugates and facilitates the excretion of sex hormones including oestrogen. Therefore, oestrogen concentrations may be elevated in female hyperbilirubinaemic rats and synergistically impact lipid metabolism [16,17]. The second study examined the effect of hyperbilirubinaemia in vitro and in vivo on mitochondrial function and body composition. Dual X-ray absorptiometry (DEXA) analysis revealed that female Gunn rats had significantly reduced fat mass (9.94 ± 5.35 vs 16.1 ± 6.65 g, P<0.05) and lean mass (140 ± 12.1 vs 160 ± 16.0 g, P<0.05) compared to littermate controls. Female Gunn rats consumed fewer calories per day (54.1 ± 6.38 vs 63.3 ± 6.95 kcal day-1, P<0.01). However, weight gain relative to calories consumed was reduced (8.09 ± 5.75 vs 14.9 ± 5.10 mg kcal-1, P<0.05) in female Gunn rats indicating that they are less energetically efficient. This led to the analysis of mitochondrial function in liver and skeletal muscle using high-resolution respirometry to ascertain the cause of reduced energetic efficiency. This analysis revealed that female Gunn rats exhibited increased oxidative phosphorylation (OXPHOS) relative to maximal noncoupled mitochondrial respiration (ETS) in hepatic mitochondria (0.78 ± 0.16 vs 0.62 ± 0.09 OXPHOS:ETS, P<0.05). The above findings were consistent with the effect of exogenous addition of unconjugated bilirubin (UCB) to control hepatic mitochondria, with 31.3 and 62.5 μM UCB increasing the OXPHOS: ETS ratio. However, exogenous UCB addition produced this effect by inhibiting ETS without affecting OXPHOS, indicating that UCB induces mitochondrial dysfunction at high concentrations. Conversely, no change in ETS (1130 ± 217 vs 1290 ± 373 pmol s-1 ng-1 citrate synthase (CS), P=0.16) or OXPHOS (901 ± 222 vs 796 ± 259 pmol s-1 ng-1 CS, P=0.36) was observed between female Gunn rats and controls. These data indicate that the greater OXPHOS:ETS ratios are a combination of increased OXPHOS and decreased ETS in female Gunn rats. Analysis of mitochondrial respiratory complexes revealed greater hepatic mitochondrial complex IV (CIV; P<0.01) expression in female Gunn rats. These findings support a conclusion that hepatic mitochondria have increased quality in female Gunn rats [18,19]. At present it remains unknown how this change in mitochondrial quality relates to reduced fat mass and energetic efficiency, however, the changes observed in female Gunn rats could represent an adaptation to bilirubin mediated inhibition of CIV as reported in vitro [20,21]. Otherwise, alterations in reproductive hormone metabolism in Gunn rats could also partially explain altered energetic states, as speculated in study one. Considering that hyperbilirubinaemia induced perturbed lipid metabolism and body composition in chapters one and two, study three sought to determine whether increasing bilirubin could alter circulating lipid profile in humans. The effect of Legalon®, containing the active ingredient silymarin, supplementation on circulating bilirubin concentrations and lipid status was investigated in a placebo controlled, single blind crossover clinical trial in healthy individuals (ACTRN12619001296123). Legalon® capsules containing 140 mg of silymarin were supplemented thrice daily (total dose of 420 mg silymarin) in a cohort of healthy males for two weeks. Two weeks of Legalon® supplementation did not change UCB concentrations compared to baseline (Legalon®: 12.5 ± 7.63 vs Baseline: 11.4 ± 4.14 μM, P=0.79). Secondary outcomes including lipid concentrations, inflammation, and total antioxidant status were also reported. Two weeks of Legalon® supplementation did not change serum cholesterol (4.80 ± 1.00 vs 4.88 ± 1.00 mM, P=0.19), triglyceride (1.07 ± 0.63 vs 1.04 ± 0.54 mM, P=0.79), C-reactive protein concentrations (1.74 ± 1.88 vs 0.92 ± 0.87 mg L-1, P=0.23) or serum antioxidant capacity (1194 ± 182 vs 1183 ± 201 mmol Fe2+ L-1, P=0.19) compared to baseline. Several clinical trials evaluating the impact of silymarin have reported changes to bilirubin concentrations following treatment [22–24]. However, these studies were conducted in patients with hepatic disease, which confounded bilirubin results, and with greater doses or different formulations of silymarin to that reported in this thesis. Although the results of this study demonstrated a negative finding, they are important because they represent the first attempt to use an orally administered, commercially approved, nutraceutical compound to increase bilirubin. These results provide important guidance to future studies that could utilise different doses or commercial preparations to induce a transient unconjugated hyperbilirubinaemia and test the impact on circulating cholesterol concentrations. In conclusion, this thesis contains three novel investigations that aimed to determine the impact of unconjugated hyperbilirubinaemia on cholesterol metabolism, synthesis and hepatic excretion; in addition to its effect on mitochondrial metabolism and body composition in Gunn rats. To determine whether these effects could be induced in humans, a nutraceutical with documented effects on circulating bilirubin was administered in a clinical trial, utilising a randomised, single-blind, crossover design. The results of this thesis suggest that bilirubin has the potential to modulate lipid and whole-body metabolism, particularly in female animals and provides the groundwork for additional studies that seek to reveal the mechanisms responsible for bilirubin’s effects. In addition, this thesis will support the discovery of more effective orally administered compounds that can modulate circulating bilirubin and lipid profile for protection against CVD.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Non-canonical bioenergetics concerns with those physiological and pathophysiological situations under which ATP synthesis is suppressed. This thesis brings an outcome of three types of studies within the field of the non-canonical bioenergetics, investigating specific bioenergetic phenotypes of cancer cells, on one hand; and a role of mitochondrial uncoupling proteins as deduced from their transcript distribution in various tissues and organs; plus a role of a novel and likely pro-apoptotic factor CIDEa in mitochondria. Cancer cells generally present abnormal bioenergetic properties including an elevated glucose uptake, a high glycolysis and a poorly efficient oxidative phosphorylation system. However, the determinants of cancer cells metabolic reprogramming remain unknown. The main question in this project was how environmental conditions in vivo can influence functioning of mitochondrial OXPHOS, because details of mitochondrial bioenergetics of cancer cells is poorly documented. We have combined two conditions, namely glucose and oxygen deprivation, to measure their potential interaction. We examined the impact of glucose deprivation and oxygen deprivation on cell survival, overall bioenergetics and OXPHOS protein expression. As a model, we have chosen a human breast carcinoma (HTB-126) and appropriate control (HTB-125) cultured cells, as large fraction of breast malignancies exhibit hypoxic tumor regions with low oxygen concentrations and poor glucose delivery. The results demonstrate that glucose presence or absence largely influence functioning of mitochochondrial oxidative phosphorylation. The level of mitochondrial respiration capacity is regulated by glucose; by Crabtree effect, by energy substrate channeling towards anabolic pathways that support cell growth and by mitochondrial biogenesis pathways. Both oxygen deprivation and glucose deprivation can remodel the OXPHOS system, albeit in opposite directions. As an adaptative response to hypoxia, glucose inhibits mitochondrial oxidative phosphorylation to the larger extent than in normoxia. We concluded that the energy profile of cancer cells can be determined by specific balance between two main environmental stresses, glucose and oxygen deprivation. Thus, variability of intratumoral environment might explain the variability of cancer cells´ bioenergetic profile. Mitochondrial uncoupling proteins are proteins of inner mitochondrial membrane that uncouple respiration from ATP synthesis by their protonophoric activity. Originally determined tissue distribution seems to be invalid, since novel findings show that UCP1 is not restricted exclusively to brown fat and that originally considered brain-specific isoforms UCP4 and UCP5 might have wider tissue distribution. Hence, in second part of this thesis, I discuss consequences of findings of UCPn transcripts in the studied mouse and rat tissues. We have shown that mRNA of UCPn varies up to four orders of magnitude in rat and mouse tissues with highest expression in rat spleen, rat and mouse lung, and rat heart. Levels of the same order of magnitude were found for UCP3 mRNA in rat 100 and mouse skeletal muscle, for UCP4 and UCP5 mRNA in mouse brain, and for UCP2 and UCP5 mRNA in mouse white adipose tissue. Further, we have shown that expression pattern of UCPn varies between animal species, rat versus mouse, such as the dominance of UCP3/UCP5 vs. UCP2 transcript in mouse heart and vice versa in rat heart; or UCP2 (UCP5) dominance in rat brain contrary to 10-fold higher UCP4 and UCP5 dominance in mouse brain. spontaneous apoptosis due to CIDEa overexpression in HeLa cells, adapted for a tetracycline-inducible CIDEa expression, a portion of mitochondria-localized CIDEa molecules migrates to cytosol or nucleus
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During the past 30-years, Biochemical System Theory (BST) has been provided a concrete foundation for the study of the dynamic biological systems, e.g. S-systems models for reverse engineering of metabolic networks (Savageau, 1969; Savageau, 1970; Voit, 2000). One of the remarkable characteristics of these models is its parameters not only quantify the interactions between the components of the network, but also elucidate the networks topology. Automatic procedures for S-system parameterization from biological time series have been developed by many researches, where they assume a noise-free time series and a true estimated first derivative in their methodologies (Chou, et al., 2006; Kikuchi, et al., 2003). Nevertheless, this noise-free data is not a realistic scenario of the real biological experimental world. Methods as artificial neural network (ANN), Support Vectors Machines (SVM) and Saviztsky-Golay filter were proposed to overcome the denoising time series problem with the advantage of a closed form output which allowed determining the first derivative symbolically (Almeida and Voit, 2003; Borges, et al., 2006; Borges, et al., 2004; Voit and Almeida, 2004). However, these solutions showed some problematic artifacts in its first derivative even when they are not visually apparent in the smoothed data, leaving a gap on the issue of a fully automatic method for S-system parameterization from experimental data. The algorithm presented in this work is a proposal to fill this gap up providing an unbiased robust tool for signal extraction and first derivative estimation from noisy time series.
Nos últimos 30 anos, a Teoria dos Sistemas Bioquímicos (Biochemical System Theory - BST) tem fornecido uma fundação concreta para o estudo da dinâmica de sistemas biológicos, por exemplo, Sistemas-S (S-systems) usados em engenharia reversa de vias metabólicas (Savageau, 1969; Savageau, 1970; Voit, 2000). Uma característica marcante desse tipo de modelo é que os parâmetros não só quantificam as interações entres os componentes da rede metabólica, mas também fornecem a sua topologia de regulação. Procedimentos automáticos para a parametrização dos Sistemas-S a partir de séries temporais biológicas vêm sendo desenvolvidos por vários pesquisadores, onde se assume que a série temporal e sua derivada temporal são livres de ruído. Entretando, perfis metabólicos livres de ruído não são um realistas em cenários de experimentos de biologia molecular. Técnicas como Redes Neurais Artificiais (RNA), Máquinas de Vetores de Suporte (MVP) e filtro de Saviztsky-Golay foram propostas como solução do problema de suavização dos perfis metabólicos com a vantagem da obtenção da derivada temporal simbólica (Almeida and Voit, 2003; Borges, et al., 2006; Borges, et al., 2004; Voit and Almeida, 2004). Entretanto, essas soluções apresentaram alguns artefatos problemáticos na derivada até mesmo quando nenhum problema é visualmente detectado no dado suavizado, deixando aberto um espaço vazio na questão de um método automático para a parametrização dos Sistemas-S a partir de dados experimentais. O algoritmo apresentado neste trabalho propõe preencher esse espaço com uma ferramenta robusta para a extração de sinal e de sua derivada temporal a partir de séries temporais ruidosas.

L'inflammation est associée à des modifications du métabolisme cellulaire avec une glycolyse (libération de lactate) accrue accompagnée d'une baisse de la phosphorylation oxydative mitochondriale. L'inflammation cause l'hyperosmolarité du milieu extracellulaire. Cette thèse examine les effets de l'hyperosmolarité sur le métabolisme énergétique cellulaire. Nous avons mesuré la consommation d'oxygène cellulaire (OCR) et la production d'acide (PPR) c'est à dire de lactate libéré dans le milieu extérieur avec deux approches expérimentales : l'oxygraphie haute résolution (O2k Oroboros instrument) pour l'OCR et l'analyseur de flux extracellulaires (Seahorse Agilent) pour l'OCR et le PPR. L'exposition de cellules à des conditions hyperosmolaires (600 mOsmoles au lieu de la valeur normale 300) cause une répression de la consommation d'oxygène qui s'établit en quelques minutes et dure des heures (indéfiniment?) et à la longue affecte la viabilité cellulaire. Cet effet a été retrouvé sur plusieurs types cellulaires: CHO (épithélium ovarien), HT29 (colonocytes), HEK293 (rein embryonnaire), SH-SY5Y (neuroblastome). Il est reproduit avec trois osmolytes différents: le mannitol, le polyéthylène glycol et le chlorure de sodium. Un stress osmotique plus modéré (450 mOsm) cause une même chute de la respiration mais de durée limitée (une-deux heures). Une recherche des mécanismes à l'origine de cette inhibition montre que l'hyperosmolarité altère la fonction mitochondriale de différentes manières. Un premier effet est une inhibition du système enzymatique de production d'ATP. En présence de glucose cette inhibition s'accompagne d'une importante augmentation de la glycolyse qui cause une inhibition mitochondriale supplémentaire qui repose sur l'amplification de l'effet Crabtree (inhibition de la respiration par le glucose) dont la cible sont les complexes respiratoires. En l'absence de glucose le turnover cellulaire e l'ATP est sérieusement diminué mais de façon inattendue la survie cellulaire est plutôt meilleure. Ces résultats posent la question de la contribution des conditions hyperosmotiques liées à l'inflammation dans l'établissement d'un profil métabolique de type inflammatoire
Metabolic alterations associated with inflammation include increased recruitment of glycolysis (lactate release) and repression of mitochondrial oxidative phosphorylation. Inflammation causes hyperosmolar conditions in the extracellular medium. This thesis examines the consequences of hyperosmolarity on cellular bioenergetics. For this purpose we measured the cellular oxygen consumption rate (OCR) and proton production rate (PPR) for lactate release in the external medium. Two methodologies were used the high-resolution respirometer (O2k Oroboros Instruments) for OCR and the extracellular flux analyzer (Seahorse, Agilent) for OCR and PPR. The exposure cells to hypertonic conditions (600 milliOsmoles while normal value is 300) causes within few minutes a decrease in OCR (cellular respiration) that lasts for hours (indefinitely) and in the long term impact on cellular viability. This effect was observed with four different cell lines CHO (ovarian epithelial), HT29 (colonocytes), HEK293 (Embryonic kidney) and SH-SY5Y (Neuroblastoma). It was shown to be caused by three different osmolytes: Mannitol, polyethylene glycol, sodium chloride. A milder osmotic challenge (450 mOsm) caused a similar initial decrease but with restoration of initial OCR within few hours. The mechanisms underlying this effect have been investigated, hyperosmolarity impacts on mitochondrial respiration at different steps. A first effect is the inhibition of the mitochondrial ATP production step. In presence of glucose this is accompanied by a large increase in glycolysis (lactate release) that causes further mitochondrial inhibition by a second mechanism, which is likely to represent an enhancement of the Crabtree effect (inhibition of respiration by glycolysis) that impacts on respiratory complexes. In absence of glucose the cellular ATP turnover is seriously repressed surprisingly cellular survival is rather improved. These results raise therefore the question of the possible contribution of the hyperosmotic conditions caused by inflammation in the acquisition of the inflammatory metabolic profile

Introduction: The reproductive senescence and the complete exhaustion of the germ cells result in processes capable of provoking changes in the hormone profile of women. The decrease in the bioenergetic metabolism during the menopause transition (MT), due to modifications in the estrogen levels, can be substrate for neurological dysfunctions. The physiopathological mechanisms of the Alzheimer’s Disease (AD) are activated years before the symptoms and coincide with MT, making the female gender a risk factor. The review aims on analyzing the higher rates of AD in the female gender, based on physiological changes that occur in the MT. Methods: Literature review based on articles from the PubMed database. Results: Were compared results from cerebral images of women in MT with cognitively normal men with the same age. In the women were found alterations such as abnormalities in the biomarkers of AD and reduction of the cerebral metabolic rate. It was noticed that women in the post menopause presented hypometabolism in the same cerebral regions as patients with AD and a reduction of the mitochondrial cytochrome oxidase of the platelets. Conclusion: The study presented evident bioenergetic factors that corroborate to the relation of MT and higher incidence of AD in the female gender. This way, such transition represents a window of opportunity for possible therapeutic interventions.

Background: Transgenic models of Alzheimer’s disease (AD) overexpress human APP, PS1 or PS2 mutations. These models present amyloid-beta pathology but do not recapitulate the complexity of AD. Interestingly, the transgenic rat model TgF344-AD, which overpresses human APP and PS1 mutations, seems to follow a more similar disease progression, manifesting progressive tau tangle-like pathology and late cognitive impairment. Yet, whether they develop energy metabolism changes as we see in AD remains unclear. Objective: Here, we investigated brain bioenergetics in 6-7 months F344-AD/WT rats, an age where animals present early amyloid pathology but no memory impairment - mimicking the human preclinical AD. Methods: We used high-resolution respirometry to assess mitochondrial oxidative phosphorylation capacity (OXPHOS), electron transfer capacity (ET), respiratory control ratio (RCR) and reserve capacity (R) in brain homogenates of male and female F344-AD and WT rats (n = 6-8, per group). Results: The results were analyzed by Welch’s t test: 1. Frontal cortex a)OXPHOS (p=0.307); b)ET (p=0.99); c)RCR (p=0.138); d)R (p=0.482). 2. Hippocampus a)OXPHOS (p=0.446); b)ET (p=0.409); c)RCR (p=0.952); d)R (p=0.503). Conclusion: In conclusion, at 6-7 months, changes in the respirometry in the brain of F344-AD rats were not observed. We hypothesize that these measures will be altered at older ages.