Dissertations / Theses on the topic 'Bioenergetic'

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

Multicellular tumour spheroids (TS) are an in vitro model of avascular tumours, and have been widely used to investigate tumour growth, metabolism and hypoxia. The geometry of the TS lends itself to mathematical representation, and theoretical models of TS growth and the development of hypoxia are abundant. With some notable exceptions however, these models have been developed independently of the biological data collection process and are overwhelmingly based upon data from multiple sources. Thus, whilst mathematical modeling has the potential to help explain and guide biological experiments, without reliable data it is unlikely to live up to this expectation. In this thesis, a combination of experimental and theoretical approaches was used to characterize the relationship between proliferation, hypoxia and metabolism during the growth of TS derived from the DLD-1 human colon adenocarcinoma cell line. Experimental data were collected over the entire period of TS growth, generating a high volume of predominantly imaging data. To facilitate the extraction of quantitative information from this, a suite of image analysis software, which is readily applicable to other data sets, was developed. During growth, the DLD-1 TS maintained a macroscopic spherical geometry but at the microscale level the TS boundary was increasingly irregular, with TS disintegrating rapidly after 20 days. Immunofluorescence (IF) studies showed that hypoxia developed soon after TS initiation, followed by the characteristic onset of necrosis. Reduced proliferation was found to be concomitant with the development of hypoxia, although some cells retained proliferative capacity even under severely hypoxic conditions. Towards the end of culture, TS were primarily comprised of severely hypoxic and necrotic cells, a probable cause of disintegration. Mathematical simulation of oxygen gradients in TS using literature-based values for the maximal rate of oxygen consumption was used to estimate the partial oxygen pressure (pO₂) at which the IF marker of hypoxia was bound. Assuming a spatially-invariant rate of oxygen consumption, the model predicted that the onset of hypoxic binding occurs at pO₂ levels similar to those reported in the literature, however the onset of necrosis was overestimated. Mathematical simulations predicted that oxygen consumption decreases as TSs increase in size, supporting previous observations. The Warburg Effect, where glucose metabolism is favoured even under aerobic conditions, is a hallmark of tumours. Although development of the glycolytic phenotype during TS growth was observed in the form of an elevated activity of the lactate dehydrogenase V (LDHV) enzyme, the activity and expression of other glycolytic enzymes, such as hexokinase II (HKII), was unaltered. Whilst the spatial distribution of HKII was unrestricted throughout the TS's viable fraction, LDHV expression was elevated in regions of hypoxia, suggesting constant adaptation of tumour cells to their microenvironment. In addition to the above findings, the data generated have been collected and analysed in the context of the requirements of theoretical modelling at each step; thus, they can be used to parameterise and inform more sophisticated models of tumour metabolism.

Migratory and resident forms of salmonid fishes, including brook trout (Salvelinus fontinalis), coexist in many river systems, but little is known about the ecological basis of these important variations in life history. This thesis elucidates the bioenergetic basis of anadromy (migration from freshwater spawning sites to the sea) in populations of brook trout. By focusing on the early stages, I provide support for the idea that variation in energy allocation leads to the adoption of migratory or resident strategies. More specifically, I demonstrate that juvenile anadromous brook trout, in the year(s) prior to migration, exhibit higher food consumption rates but lower growth efficiencies compared to residents indicating that they have higher metabolic costs. Higher metabolic costs of migratory fish are associated with the exploitation of higher current velocity habitats that provide more food but at a higher cost. This conclusion is supported by differences in delta13C (migrants have more negative delta13 C compared to residents), morphology (migrants are more streamlined than residents), and field observations (brook trout inhabiting streams with both forms exploit a wider range of habitats than those inhabiting 'pure' resident streams). Brook trout thus appear to migrate in response to energetic limitations in their local habitat. The estuary to which they migrate has better feeding opportunities, as the prey spectrum at sea is both larger and wider than that found in freshwater. This permits them to undergo diet shifts to larger prey, reducing their foraging costs, and thus most likely contributes to the trout's rapid growth rates experienced at sea. Importantly, the results of this thesis indicate that the persistence of migrant and resident strategies in the same system suggest a trade-off between local adaptability and the ability to exploit large-scale environmental heterogeneity.

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.

After advanced age, female sex is the major risk factor for Alzheimer's disease (AD). The biological mechanisms underlying the increased AD risk in women remain largely undetermined. Preclinical studies identified the perimenopause to menopause transition, a neuroendocrine transition state unique to the female, as a sex-specific risk factor for AD. In animals, estrogenic regulation of cerebral glucose metabolism (CMRglc) falters during perimenopause. This is evident in glucose hypometabolism and decline in mitochondrial efficiency which is sustained thereafter. This study bridges basic to clinical science to characterize brain bioenergetics in a cohort of forty-three, 40-60 year-old clinically and cognitively normal women at different endocrine transition stages including premenopause (controls, CNT, n = 15), perimenopause (PERI, n = 14) and postmenopause (MENO, n = 14). All participants received clinical, laboratory and neuropsychological examinations, F-18-fluoro-deoxyglucose (FDG)Positron Emission Tomography (PET) FDG-PET scans to estimate CMRglc, and platelet mitochondrial cytochrome oxidase (COX) activity measures. Statistical parametric mapping and multiple regression models were used to examine clinical, CMRglc and COX data across groups. As expected, the MENO group was older than PERI and controls. Groups were otherwise comparable for clinical measures and distribution of APOE4 genotype. Both MENO and PERI groups exhibited reduced CMRglc in AD-vulnerable regions which was correlated with decline in mitochondrial COX activity compared to CNT (p's<0.001). A gradient in biomarker abnormalities was most pronounced in MENO, intermediate in PERI, and lowest in CNT (p<0.001). Biomarkers correlated with immediate and delayed memory scores (Pearson's 0.26 <= r <= 0.32, p <= 0.05). These findings validate earlier preclinical findings and indicate emergence of bioenergetic deficits in perimenopausal and postmenopausal women, suggesting that the optimal window of opportunity for therapeutic intervention in women is early in the endocrine aging process.

Black carp consume large quantities of mollusks but food consumption and growth rates have not been studied. Consumption and growth of two sizes of fish fed formulated and natural diets at 20, 25, and 30 C under laboratory conditions were measured. Consumption and growth of fish fed formulated feed increased as temperature increased but followed a convex relationship for fish fed a natural diet. Metabolic and fecal production rates were determined for fish at each temperature. This information was used to develop balanced energy budgets for black carp. Bioenergetic simulation models were constructed to predict consumption and growth in natural systems. Fish inhabiting the Tennessee River and Lake Erie grow faster than fish in other select natural systems of the eastern United States and probably provide a highly satisfactory thermal regime.

This PhD thesis describes the effects of Vitamin D on skeletal muscle and includes details of two main research projects. The first study validated a physiologically significant effect of VitD3 in proliferation, differentiation, protein synthesis and mitochondrial metabolism in primary human skeletal muscle cells, in cell culture. The second study demonstrated that VitD3 supplementation does not result in additional benefits on muscle strength, power, resting metabolic rate and body composition in VitD sufficient physically active adults.

Las leishmaniasis, la tripanosomiasis Americana y Africana, y la malaria son enfermedades parasitarias que constituyen un importante problema de salud global que afecta mayoritariamente a países en desarrollo. El aumento del número de resistencias a sus tratamientos actuales, su toxicidad y la necesidad de asistencia sanitaria para la aplicación de los mismos reflejan la urgente necesidad de desarrollar vacunas eficaces y nuevos tratamientos económicos, fáciles de administrar y resistentes a condiciones de almacenamiento adversas. Basándonos en que estas enfermedades parasitarias comparten requerimientos metabólicos con patologías mejor estudiadas, proponemos el reposicionamiento de fármacos para tratarlas. Bajo esta premisa, seis fármacos de eficacia probada en la investigación contra el cáncer ―dicloroacetato (DCA), 3‐bromopiruvato (3BP), 2‐ deoxi‐D‐glucosa (2DG), lonidamina (LND), metformina (MET) y sirolimus (SIR)― fueron seleccionados por su habilidad para modular rutas metabólicas relacionadas con la producción de energía y proliferación. El objetivo de este estudio fue validar el uso de estos moduladores bioenergéticos para el control de la leishmaniasis visceral, malaria y tripanosomiasis americana y africana como tratamiento o como potenciadores de la protección de una vacuna frente a L. infantum. Para ello, se evaluó la eficacia de estos compuestos en modelos in vitro de cada parasito ―enfermedad de Chagas (Trypanosoma cruzi), tripanosomiasis Africana (Trypanosoma brucei), leishmaniasis visceral (Leishmania infantum) y malaria (Plasmodium falciparum)―. El 3BP y el DCA indujeron una reducción dosis‐dependiente del crecimiento de los amastigotes intracelulares de L. infantum con IC50 de 17.19 μM y 631.5 μM, respectivamente. En el modelo in vitro de T. brucei, todos los compuestos testados, a excepción de 2DG, afectaron a la viabilidad del parásito: DCA (IC50 = 1.24 mM), 3BP (IC50 = 76.57 μM), LND (IC50 = 26.76 μM), SIR (IC50 = 2.14 μM), y MET (IC50 = 17.30 Mm). En el caso de los amastigotes intracelulares de T. cruzi, DCA, 3BP, 2DG, LND, y MET tuvieron efecto parasiticida con valores de IC50 de 27.07 mM, 27.63 μM, 7.27 mM, 78.37 μM, y 18.48 mM, respectivamente. DCA (IC50 = 5.39 mM), 2DG (IC50 = 4.19 mM), LND (IC50 = 209.13 μM), MET (IC50 = 1.32 mM), y SIR (IC50 = 2.50 μM), mostraron efecto antiparasitario sobre trofozoitos de P. falciparum. Estos resultados sugieren que estos fármacos podrían ser útiles para tratar estas enfermedades parasitarias. Sin embargo, cuando los compuestos eficaces en los modelos in vitro fueron administrados en modelos in vivo de roedor para cada una de las enfermedades, ninguno de ellos contribuyó al control de la enfermedad o de la carga parasitaria. Los resultados obtenidos en el modelo de leishmaniasis visceral en hámster revelaron una disminución de la activación del sistema inmune en los animales tratados con DCA y 3BP, lo cual podría haber contribuido al fracaso del tratamiento. Por último, se estudió la capacidad del SIR para potenciar el efecto protector de una vacuna frente a la leishmaniasis visceral en el modelo hámster. Para ello se administró SIR durante la fase de expansión y contracción del sistema inmune producido por una vacuna de DNA portadora de los genes LACK, TRYP, PAPLE22, y KMPII de Leishmania, y se estudió la respuesta frente al posterior desafío con L. infantum. Los resultados muestran que la vacuna de DNA indujo la reducción eficaz de la carga parasitaria en piel (P = 0.0004) y linfonodos (P = 0.0452), lo cual potenció la administración del SIR alcanzándose también protección parasitológica en bazo (P = 0.0004). El estudio de los marcadores inmunológicos en dicho órgano sugiere que la producción controlada de IFN‐γ y el incremento en la expresión de FoxP3 podrían ser los responsables de la protección alcanzada.
Leishmaniases, African and American trypanosomiases and malaria are parasitic diseases that constitute a major global health problem. The increasing number of drug‐resistances to their current treatments, toxicity cases and the health assistance often required for their administration, makes it urgently necessary to develop efficient vaccines for humans and new affordable therapies, easy to apply and resistant to harsh storage conditions. Due to the fact that these diseases share similar metabolic requirements with better studied diseases, we chose drug repurposing as a potentially effective approach against them. With this purpose, six different compounds used in anti‐cancer research —dichloroacetate (DCA), 3‐bromopyruvate (3BP), 2‐deoxy‐D‐glucose (2DG), lonidamine (LND), metformin (MET), and sirolimus (SIR)— were selected according to their ability to modulate energy production and proliferation related metabolic pathways. The aim of this study was to validate the suitability of these bioenergetics modulators for the management of visceral leishmaniasis, malaria and African and American trypanosomiasis as a treatment, or as a preventive tool by enhancing the protective power of a vaccine against L. infantum. The effectiveness of these compounds was first evaluated on in vitro models of each parasite ― Chagas disease (Trypanosoma cruzi), human African trypanosomiasis (Trypanosoma brucei), visceral leishmaniasis (Leishmania infantum) and malaria (Plasmodium falciparum)―. L. infantum promastigotes were not susceptible to these compounds, whereas L. infantum intracellular amastigote growth was dose‐dependently reduced by 3BP (IC50 = 17.19 μM) and DCA (IC50 = 631.5 μM). In the T. brucei in vitro model all the tested compounds, with the exception of 2DG, affected parasite survival with IC50 values of 1.24 mM for DCA, 76.57 μM for 3BP, 26.76 μM for LND, 2.14 μM for SIR, and 17.30 mM for MET. In the case of T. cruzi, DCA, 3BP, 2DG, LND, and MET showed parasite‐killing activity with IC50 values of 27.07 mM, 27.63 μM, 7.27 mM, 78.37 μM, and 18.48 mM, respectively. For P. falciparum DCA (IC50 = 5.39 mM), 2DG (IC50 = 4.19 mM), LND (IC50 = 209.13 μM), MET (IC50 = 1.32 mM), and SIR (IC50 = 2.50 μM), showed antiplasmodial activity. These results reinforce the hypothesis that drugs with proven efficacy in the treatment of cancer by interfering with energy production might be useful in treating threatening parasitic diseases and provide new opportunities for their repurposing. However, when compounds that were effective in the in vitro approach were administered to the in vivo rodent models of these diseases, none of them contributed to disease management or parasite load control. Immunological analysis in the VL hamster model revealed a significant downregulation of immune‐activation in infected animals treated with DCA and 3BP, which may also contribute to treatment failure. In the last chapter of this work, the suitability of sirolimus as an immunomodulatory compound to boost the activity of a preventive vaccine against VL was analyzed. Sirolimus is an already marketed compound that has been described to boost immune protection against different disease models. In our study, Syrian hamsters were treated with sirolimus concomitantly with the administration of a plasmid DNA vaccine carrying the Leishmania genes LACK, TRYP, PAPLE22 and KMPII, and the subsequent response towards a L. infantum challenge was studied. Our results show that the DNA vaccine itself efficiently reduced the burden of parasites in skin (P = 0.0004) and lymph nodes (P = 0.0452), which was potentiated by SIR administration by also inducing parasitological protection in the spleen (P = 0.0004). The study of immune markers in spleen suggests that lower production of IFN‐γ and the concurrent increase of FoxP3+ expression may be responsible for the protection mediated by the DNA vaccine that was potentiated by sirolimus.

During early lactation dairy cows often experience health disorders, which are usually associated with decreased production and reproduction variables. Following parturition, cows use more energy for maintenance and milk production than they consume and enter into a state of negative energy balance. Negative energy balance in early lactation is thought to contribute to decreased milk production, reduced reproductive performance, and increased health disorders. Flunixin meglumine (FM) is an anti-pyretic (fever reducing) and anti-inflammatory drug that is commonly used in the dairy industry. This study evaluated the effect of FM on pyrexia, production and bioenergetic variables in postparturient dairy cows.

Animal behaviour is a state variable of the individual that deserves special attention given its determinant role in eco-evolutionary processes (Wolf et al. 2007 in Nature). The decomposition of the behavioural variation in between- and within-individual variability has revealed the existence of consistent between-individual differences referred to as personality or behavioural types (Dall et al. 2004 in Ecology Letters). Five axes of personality are usually recognized (exploration, aggressiveness, activity, sociability and boldness), and individual specificities along them tend to be correlated leading to what is known as behavioural syndromes. Recently, these patterns of covariation have been enlarged to accommodate movement behaviour within a personality-dependent spatial ecology theory (Spiegel et al. 2017 in Ecology Letters). Most animals tend to forage, reproduce and develop any activity within specific bounded space, which leads to the formation of home range (HR) areas (i.e., HR behaviour, Börger et al. 2008 in Ecology Letters). The increasing development of animal tracking technology is providing a huge amount of movement data revealing that HR behaviour is widespread among taxa and shows a large consistent variability, both at within- and between-individual level, which allows to define the existence of well-contrasted spatial behavioural types (SBTs). SBTs, as other personality traits, play an important role in selective processes as those impelled by harvesting activities. The Pace-of-Life-Syndrome (POLS) theory (Réale et al. 2010 in Philos. Trans. R. Soc. B Biol. Sci), hypothesises on how personality traits are expected to be correlated with life history (LH) traits along the fast-slow continuum (Stearns 1992 in Oxford Univ. Press) in the broadest sense. Accordingly, patterns of covariation between specific SBTs, physiology-related features and LHs would be expected to exist whenever they maximize the animal performance in a given environment. However, the way in which behavioural variation at the within-species level is translated to the wide range of LH traits remains a fundamental yet unresolved question, mainly due to the lack of a proper theoretical framework (Mathot & Frankenhuis, March 2018 in Behavioral Ecology and Sociobiology). Thus, unrevealing the mechanisms behind is certainly scientifically very exciting but also socially relevant. In such a context, this PhD thesis aimed to address from conceptual, empirical and theoretical perspectives cornerstone questions in behavioural ecology: what are the feasible mechanisms underpinning the establishment of HR areas and within-species variation, what are their consequences for animal functioning and performance (i.e., in. LH traits) at the individual and eco-evolutionary levels, or what are the implications for the assessment and conservation of wildlife of the existence of SBTs. The PhD thesis focusses in a fish heavily exploited by recreational fishers but it aims to provide general reasoning applicable to a wide range of wild animals. First, the PhD thesis proposes a mechanistic theory of personality-dependent movement behaviour based on dynamic energy budget models (i.e., a behavioural-bioenergetics theoretical model). Second, integrated in the field of animal personality (i.e., decomposition of behavioural variability into within- and between-individual’s components), it addresses empirically the study of behavioural variability in the main axis of personality for a marine fish species and looked for evidences of whether personality-mediated differences in energy acquisition may exist. Aiming to support empirically the possible connections between personality traits and space-use behaviour, the thesis provides some insights on the application of a novel-tracking algorithm to analyse the movement of individual fish submitted to different experimental conditions. Third, it provides two examples of how applying HR-related theoretical concepts may improve the management of natural resources: attending the properties of HR may facilitate the assessment of wildlife using fixed monitoring sampling stations, and considering SBTs may influence the assessment of the status of wild fish stocks. Finally, the adaptive value of the proposed behavioural-bioenergetics theory is explored by means of dynamic optimization to understand the eco-evolutionary consequences related with HR variability. In summary, this PhD thesis makes an important contribution to behavioural ecology by developing a unifying theory to test the generality and adaptive value of POLS based on dynamic energy budgets. This behavioural-bioenergetics model connects (1) personality traits (2) HR behaviour, (3) physiology and (4) LH traits through an interwoven of mass/energy fluxes, within which they interact and feedback with the ecological context. Overall, from an eco-evolutionary perspective, the proposed framework constitutes a powerful tool for exploring the ecological role of HR behaviour and predicting what combination of behavioural traits would be evolutionally favoured in a given ecological context. Moving forward to including managerial scenarios, this unifying theory provides scientifically founded knowledge that would promote to improve natural resource management by attending the behavioural component of animal populations.

Le but d’un traitement anticancéreux est d’être spécifique et efficacité dans la durée vis-à-vis des cellules tumorales. De nombreux agents chimiothérapeutiques ont rencontré des obstacles quant à leur utilisation en raison de leur toxicité pour les cellules saines ou de la résistance développée par les cellules cancéreuses. Cela souligne la nécessité de développer des médicaments alternatifs. Notre laboratoire a développé une classe d'inhibiteurs de réparation de l'ADN, Dbait, qui agissent en détournant et en hyperactivant les protéines de la réparation de l’ADN, telles que la protéine PARP et DNA-PK. Cela conduit en conséquence à des modifications de la chromatine, visualisées par la phosphorylation pan-nucléaire de l’histone H2AX, et en l’inhibition du recrutement aux sites des dommages de plusieurs protéines de réparation. AsiDNA, une forme active de Dbait, sensibilise les tumeurs aux radiations, à la chimiothérapie, à la thérapie ciblée, sans effet sur les cellules non tumorales et les tissus sains. Dans la mesure où la chimiothérapie consiste en des traitements cycliques de l'agent anti-cancéreux, l'objectif de cette étude était d'étudier in vitro les conséquences d’un traitement répété d’AsiDNA sur les cellules tumorales et non tumorales, plus particulièrement pour ce qui concerne l’émergence de clones tumoraux résistants ou inversement de clones non tumoraux devenus sensibles au traitement. Dans un premier temps, nous avons conçu des expériences dans le but d'isoler des clones résistants au traitement par AsiDNA. Nous montrons que des traitements cycliques ne conduisent pas à des clones résistants, mais au contraire à la sélection de cellules tumorales caractérisées par une hyper sensibilité à l'AsiDNA. Cette sensibilité acquise est stable dans le temps et n'a jamais été observée en traitant des cellules non tumorales. Afin d’identifier le(s) mécanisme(s) responsable(s) de cette sensibilité acquise, nous avons comparé des cellules de sein non tumorales (MCF-10A) et tumorales triples négatives (MDA-MB-231) après 3 trois cycles de traitement par AsiDNA. Nous montrons que les traitements cycliques d'AsiDNA causent une inhibition de l'expression génique, essentiellement au niveau de gènes impliqués dans la réparation de l'ADN, le cycle cellulaire et la prolifération. Néanmoins, aucune différence dans la capacité de réparation de l'ADN, la progression du cycle cellulaire et le taux de prolifération n'est observable. Les cellules cancéreuses augmentent les voies métaboliques énergétiques pour produire d’énergie nécessaire à leur prolifération. En tenant compte du fait que l’expression de certains gènes impliqués dans les voies métaboliques sont aussi dérégulées par le traitement cyclique d’AsiDNA, nous avons émis l’hypothèse que l’épuisement métabolique pouvait être responsable de la sensibilisation des cellules tumorales à l’AsiDNA. Une étude du métabolome a révélé une dérégulation de plusieurs métabolites incluant NAD+. Nous montrons que cette dérégulation bioénergétique est responsable de l'hypersensibilité acquise des cellules cancéreuses suite au traitement par AsiDNA. Une étude bioénergétique des cellules tumorales non traitées et sélectionnées après les traitements cycliques par AsiDNA confirment une diminution de glycolyse aérobique et de la phosphorylation oxydative dans ces dernières. En conséquence de cette réduction énergétique, les cellules cancéreuses ont perdu leur caractère malin, ce qui est démontré par une inhibition de la migration et de la formation de tumeur. Nous montrons que les cellules tumorales dérivées de traitements cycliques par AsiDNA sont dépourvues de cellules souches cancéreuses dont les caractéristiques sont leur résistance aux drogues et leur phénotype invasif. En conclusion, à côté de son rôle dans l'inhibition de la réparation de l'ADN, AsiDNA interfère également avec le métabolisme énergétique des cellules cancéreuses
The goal of anti-cancer treatment is long term specificity and efficacy towards cancer cells. Many of the clinically available chemotherapy have encountered obstacles due to their toxicity towards healthy cells or to development of resistance by the cancer cells. This emphasizes the need for development of alternative drugs. Our laboratory developed an original class of DNA repair inhibitor, Dbait, that acts by hijacking and hyper activating DNA repair proteins involved in repairing DNA breaks, such as PARP and DNA-PK. Consequently, this leads to chromatin modification, as revealed by pan-nuclear phosphorylation of H2AX, and inhibition of the recruitment at the damage site of several DNA repair proteins at the damage site. AsiDNA, an active form of Dbait linked to a cholesterol moiety, sensitizes tumours, and not non-tumour cells, to radiation, chemotherapy, targeted therapy. As most of clinical protocols of chemotherapy involve cyclic treatments, the aim of this study was to investigate consequences of cyclic AsiDNA treatment in vitro on non-tumor and tumor cells, conditions that experience cancer patients during chemotherapy. Particular emphasis was paid to emergence of resistant clones during cyclic AsiDNA treatment of tumour cells and emergence of toxicity toward normal cells. At first, various tumor and non-tumor cells were exposed to cyclic treatments consisting of one week of treatment and one week of drug-free recovery. After few cycles of treatment, we didn’t observe toxicity toward normal cells and we failed to isolate resistant clones to AsiDNA from tumor cells. Importantly, this treatment protocol induced resistance of MDA-MB-231 cells to imatinib or PARPi. Unexpectedly, we observed that sensitivity to AsiDNA increased with repeated cycles in tumor cells. This acquired sensitization was stable over time and was never observed in non-tumor cells. In an attempt to understand the specific and acquired sensitization of tumor cells along treatment, we compared non-tumor (MCF-10A) and triple-negative breast cancer (MDA-MB-231) cells that were exposed (3CAsiDNA) or not (3CMT) to 3 rounds of AsiDNA. Transcriptome analysis of MDA-MB-231 revealed global downregulation of transcription after cyclic AsiDNA treatment. Although the expression of genes involved in DNA repair, cell cycle and proliferation, was highly affected, strikingly no clear difference in DNA repair capacity, cell cycle or proliferation rate was observed between MDA-MB-231_3CAsiDNA and MDA-MB-231_3CMT. In contrary, modification of gene expression was weakly affected in non-tumor cells.As impaired DNA repair capacity or cell cycle deregulation couldn’t explain this acquired sensitivity, therefore alternative mechanisms should account for the higher mortality of cyclic treated AsiDNA cells. Cancer cells upregulate energy metabolic pathways to produce enough energy for cell proliferation and repair. Noteworthy, AsiDNA is a PARP activator requiring NAD+ consumption. Based on the fact that metabolic pathways were also deregulated at the transcriptional level, we hypothesized that metabolic exhaustion may be responsible for AsiDNA induced sensitization. Metabolome study revealed deregulation of several metabolites including NAD+. We showed that this bioenergetics deregulation is responsible for increasing sensitivity to AsiDNA. Bioenergetics study confirmed low metabolic activity after repeated AsiDNA treatment due to deregulating aerobic glycolysis and oxidative phosphorylation. As a consequence of energetic deprivation, cancer cells deregulated their malignant behavior by inhibition of migration and tumor formation. We showed that 3CAsiDNA tumor cells are depleted of cancer stem cells, which features are responsible of drug resistance and cancer invasive phenotype. Altogether, we demonstrated that AsiDNA, beside its role in DNA repair inhibition, also interferes with energy metabolism in cancer cells

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Bioenergetic Body Therapy, created by Lowen and Pierrakos, has been used since the 1950s to help people express pain, traumatic experience, emotional conflict and pleasure. It defends the idea that the human being is not only their body but also their thoughts, emotions, sensations and actions. This research is a study of mixed methods, making use of a research approach that associates the quantitative and qualitative forms with multiple case studies, in the light of Bowlby's Attachment Theory and Bioenergetic Analysis that descends from the discoveries and theories of Wilhelm Reich and Freud's Psychoanalysis. The objective of this research, therefore, is to investigate the effects of the psychotherapeutic action obtained by means of the Bioenergetic Analysis with bereaved people; to analyze the results of the psychotherapeutic action, considering the factors of protection and the factors of risk present in the grieving process. The participants were eight adults of varying ages who were in bereavement for having lost their beloved ones due to various causes. The participants were divided into two groups: Experimental Group – EG, which took part of 14 sessions of psychotherapy for six months, and the Control Group – CG, which remained on the waitlist for six months. The research instruments employed were: clinical interview (before and after the interventions), the Hogan Grief Reaction Checklist measuring instrument – HGRC (before and after the interventions), exercises and body experiences. Case studies were carried out, articulating the data observed during the body work to the results obtained through the HGRC and the interviews. The results of the Hogan Grief Reaction Checklist measuring instrument – HGRC showed statistically significant improvements in the EG in the six subscales: despair, panic, personal growth, guilt and anger, detachment and disorganization. The CG showed improvements only in personal growth. The results indicate that the effects of the psychotherapeutic action obtained through Bioenergetic Analysis favored the participants of this research in their grieving process
A Terapia Corporal Bioenergética, criada por Lowen e Pierrakos, tem sido utilizada desde os anos 1950 com o intuito de ajudar as pessoas a expressarem dor, experiência traumática, conflitos emocionais e prazer. Defende a ideia de que o ser humano é seu corpo como também seus pensamentos, emoções, sensações e ações. A presente pesquisa é um estudo de métodos mistos, fazendo uso de uma abordagem de investigação que associa as formas quantitativa e qualitativa, com estudos de casos múltiplos, à luz da Teoria do Apego de Bowlby e da Análise Bioenergética que descende das descobertas e teorias de Wilhelm Reich e da Psicanálise de Freud. O objetivo desta pesquisa, portanto, consiste em investigar os efeitos da ação psicoterapêutica obtidas por meio da Análise Bioenergética com pessoas enlutadas; analisar os resultados da ação psicoterapêutica, considerando os fatores de proteção e os fatores de risco presentes no processo de luto. Os participantes foram oito pessoas adultas enlutadas com idades variadas que tinham perdido entes queridos por causas diversas. Foram divididos em dois grupos: Grupo Experimental – GE, que recebeu 14 sessões de psicoterapia durante seis meses e o Grupo Controle – GC, que ficou em lista de espera durante seis meses. Os instrumentos de investigação utilizados foram: entrevista clínica (antes e após das intervenções), instrumento de mensuração Hogan Grief Reaction Checklist – HGRC (antes e após das intervenções), exercícios e vivências corporais. Foram realizados estudos de caso, articulando o observado no trabalho corporal aos resultados obtidos no HGRC e na entrevista. Os resultados do instrumento de mensuração Hogan Grief Reaction Checklist – HGRC apresentaram melhoras estatisticamente significativas no GE nas seis subescalas: desespero, pânico, crescimento pessoal, culpa e raiva, desapego e desorganização. O GC apresentou melhoras apenas no crescimento pessoal. Os resultados indicam que os efeitos da ação psicoterapêutica obtidos por meio da Análise Bioenergética favoreceram os participantes desta pesquisa no seu processo de luto

Thesis (Ph. D.) -- University of Maryland, College Park, 2008.
Thesis research directed by: Marine, Estuarine, Environmental Sciences Graduate Program. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Thesis (M.S.) -- University of Maryland, College Park, 2009.
Thesis research directed by: Marine, Estuarine, Environmental Sciences Graduate Program. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

The population of harbour ragworms, Nereis diversicolor, inhabiting the upper reaches of Restronguet Creek in the Fal Ria, UK, are known to be resistant to the acute, toxic effects of copper, zinc and cadmium. This research aimed to establish whether metabolic and reproductive performance costs were associated with the resistance phenomenon and what the biochemical mechanisms of resistance were, by comparing animals from the Creek with two comparable study sites in the South West of England: Froe Creek and the Teign estuary. There was a significant metabolic cost, measured as a reduction in Scope for Growth, associated with the resistance phenomenon. As no differences in energy intake or uptake were found between resistant and non-resistant animals it was concluded that this cost was covered by demand-side regulation of energetics. A reduction in the amount of biochemical energy reserves in the form of lipids and sugars was also observed in resistant animals suggesting that resource allocation had been shifted away from this endpoint and towards maintenance and activity. The perturbed metabolism and physiology of resistant N. diversicolor is shown to have detrimental consequences for their life-history. Sexually mature, resistant females were significantly smaller than non-resistant ones, indicating that they had either matured at a younger age or grown more slowly. Both total fecundity and mass-specific fecundity were significantly reduced in resistant females, likely as a direct result of the metabolic costs of resistance reducing the resources available to fuel gametogenesis. No differences were found in the energetic reserves stocked within each gamete by resistant and non-resistant animals, suggesting that this trait is heavily selected for. Synthesis of reduced glutathione [GSH] in resistant animals’ tissues appeared to be elevated. Glutathione peroxidase activity was also increased, likely to remediate the effects of Reactive Chemical Species [RCS] that result from the inevitably incomplete binding of intracellular metals. As GSH functions in metal detoxification to covalently binding metals entering resistant animals’ cells, preventing their involvement in toxic interactions and their catalysis of the production of RCS, it is proposed that together there two phenomena comprise the fundamental mechanism of resistance to metal toxicity. Ultimately, this research revealed a hierarchy of health and reproductive performance across the three study populations, with significant associations evident between measured biological endpoints and the degree of metal contamination, illustrating the consequences of anthropogenic pollution for the biology of wild animals.

Thesis (Ph. D.) -- University of Maryland, College Park, 2008.
Thesis research directed by: Marine, Estuarine, Environmental Sciences Graduate Program. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Multidrug efflux pump P-glycoprotein (P-gp) has been the subject of significant research interest since the discovery of the protein in multi-drug resistant cancer cells 40 years ago. Overexpression of P-gp can confer resistance to a broad range of structurally and functionally distinct drugs. The mechanism of this polyspecificity is poorly understood, yet the mechanism is known to involve a complex series of binding, hydrolysis and dissociation events that are coupled to an array of conformational changes. A wealth of biochemical data implicates TM6 & 12 as mediators of bioenergetic coupling in P-gp. A major issue in attempts to determine the molecular mechanism is the lack of a high-resolution structure of human P-gp in the basal, inwardfacing state. Several crystal structures of murine P-gp in the basal state are available, and molecular dynamics have been used to simulate the structure of human P-gp. Both of these structural models are subject to controversy. Crystallisation requires solubilisation of P-gp into a detergent micelle, an environment in which the protein has been demonstrated to be inactive; the crystal structures published to date also differ significantly. MD simulations are an increasingly powerful tool for predicting protein structure; however, these must be treated with caution when not supported by experimental data. The present investigation determined the relative position of two transmembrane helices, six and twelve (TM6 & 12), by measuring the distances between residues. Site directed mutagenesis was used to generate nine double mutants, each containing one cysteine on TM6 and one on TM12. Double mutants were expressed using the baculovirus/insect cell expression system. Crude membranes containing P-gp were isolated, followed by purification by affinity chromatography. P-gp mutants were reconstituted into proteoliposomes; retention of function in the P-gp double mutants was confirmed using ATP hydrolysis assays. Inter-residue distance measurements were performed using intramolecular cross-linking with homobifunctional reagents and 5 double resonance EPR. Further ATP hydrolysis assays were used to determine the effect of intramolecular cross-linking between TM6 & 12 on the activity of P-gp. All the double mutants were functional, and most inter-residue distances were measured successfully by both techniques. The topographical map generated was compared to distance measurements in the crystal structures and MD simulations to judge whether either of these models are a good representation of human P-gp in a lipid bilayer. It was found that the 4M1M structure of murine P-gp is the most accurate representation among models derived by crystallography, while MD simulations based on this are closer still to experimental observations. The experimental results strongly suggest that the side chain orientations are accurate in the models in the membrane embedded portion of TM6 & 12. This investigation therefore demonstrated that 4M1Mbased structural models can be relied upon in the important membrane-integral region, which has been shown to be the site of drug binding. This can give greater weight to in silico methods for predicting molecular interactions with P-gp. Further details of the role of the two helices in the P-gp mechanism were added by analysis of functional changes in P-gp containing intramolecular cross-links between TM6 & 12. Specifically, the results presented here indicate that different parts of the helices require relative movement during drug-stimulated ATP hydrolysis in comparison to basal hydrolysis, suggesting that the conformational transitions of P-gp may be different in the two cases.

Orientador: Pedro Luiz Onofrio Volpe
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica
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Mestrado
Físico-Química
Mestre em Ciências

Background: Adipose-derived stem/stromal cells (ASCs) isolated from the stromal vascular fraction are a source of mesenchymal stem cells that have been shown to be beneficial in many regenerative medicine applications. ASCs are an attractive source of stem cells in particular, due to their lack of immunogenicity. This study examines differences between mitochondrial bioenergetic profiles of ASCs isolated from adipose tissue of five peri-organ regions: pericardial, thymic, knee, shoulder, and abdomen. Results: Flow cytometry showed that the majority of each ASC population isolated from the adipose tissue of 12 donors, with an n = 3 for each tissue type, were positive for MSC markers CD90, CD73, and CD105, and negative for hematopoietic markers CD34, CD11B, CD19, and CD45. Bioenergetic profiles were obtained for ASCs with an n = 4 for each tissue type and graphed together for comparison. Mitochondrial stress tests provided the following measurements: basal respiration rate (measured as oxygen consumption rate [pmol O-2/min], ATP production, proton leak, maximal respiration, respiratory control ratio, coupling efficiency, and non-mitochondrial respiration. Glycolytic stress tests provided the following measurements: basal glycolysis rate (measured as extracellular acidification rate [mpH/min]), glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. Conclusions: The main goal of this manuscript was to provide baseline reference values for future experiments and to compare bioenergetic potentials of ASCs isolated from adipose tissue harvested from different anatomical locations. Through an investigation of mitochondrial respiration and glycolysis, it was demonstrated that bioenergetic profiles do not significantly differ by region due to depot-dependent and donor-dependent variability. Thus, although the physiological function, microenvironment and anatomical harvest site may directly affect the characteristics of ASCs isolated from different organ regions, the ultimate utility of ASCs remains independent of the anatomical harvest site.

A bioenergetics model was constructed for stream-resident drift-feeding salmonids. Model predictions of surplus power (energy available per unit time for lll growth and reproduction) were not statistically distinguishable from observations of surplus power in three laboratory studies. Of 40 experimental trials in these three studies, the model correctly predicted surplus power in 39 cases (p < 0.05). I collected observations of rainbow trout (Oncorhynchus mykiss) focal velocity and physical habitat availability in the Green River of northeastern Utah, USA (1988-1990). In the winter of 1988, Flaming Gorge Dam generated hydropower and delivered an lJDStable discharge regime with a higher mean discharge to the Green River. During 1989 and 1990, Flaming Gorge Dam's operation was curtailed by drought. Therefore, the Green River exhibited a more stable discharge regime with lower mean daily discharge. During winters exhibiting the stable discharge regime, all size classes of rainbow trout selected slower focal velocities than under an unstable winter discharge regime. Season had less influence on microhabitat selection of large fish than smaller individuals. Rainbow trout larger than 33 cm (total length) find and use positions with low focal velocities and high velocity shear regardless of season. In contrast, during the summer, fish less than 33 cm TL find and use positions with much higher focal velocities and greater velocity shear compared to the winter. Four bioenergetic models were tested with the focal velocity use data. Two optimal goal models produced excellent fits (r2 = 0.91 and 0.93) to observed focal velocity use of rainbow trout larger than 33 cm TL. These results were consistent with the hypothesis that large rainbow trout were finding optimal focal velocity positions in stable discharge summers and under both discharge regimes in winter. Rainbow trout movement was quantified along two scales with radio-telemetered fish: 1) weekly observations generated estimates of distances moved at intervals greater than one day and 2) multiple observations of a fish in one day produced estimates of distances moved over hours. I found an unstable discharge regime significantly reduces movement measured weekly (F = 11.10, P = 0.0019); hourly movement rates (m/h) were also reduced (F = 5.90, P = 0.0273).

Lo squilibrio tra produzione di ROS e loro eliminazione porta a stress ossidativo, una condizione implicata nella patogenesi di molti disordini cellulari come la neurodegenerazione e il cancro. In questi constesti, condizioni pro-ossidanti attivano molte proteine redox-sensibili coinvolte nella regolazione del programma apoptotico. Tra di questi, p53 e le MAPK sono implicate nella patogenesi dei disordini neurodegenerativi e in molte strategie chemioterapeutiche finalizzate alla eliminazione di cellule cancerose dal pool replicativo. Recentemente AMPK è stata identificata come un componente della cascata di segnalazione capace di percepire stress bioenergetici e ossidativi. Inoltre la sua capacità di indurre apoptosi attivando p53 o taluni membri della famiglia delle MAPK (ad es. p38MAPK), la rende implicata sia nelle malattie neurodegnerative che nel trattamento del cancro. Sulla base di queste conoscenze, questa tesi di dottorato è finalizzata ad elucidare le prpoprietà pro-apoptotiche di AMPK nella linea neuronale SH-SY5Y in risposta a condizioni pro-ossidanti di AMPK associate a stress bioenergetico. La ricerca è stata realizzata usando 3 composti noti influenzare l'omeosatsi redox:Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N’]copper(II) perchlorate (Cu(isaepy)2), tetrahydrobiopterin (BH4) and sodium nitroprusside (SNP).
The imbalance between ROS production and clearance leads to oxidative stress, a condition implicated in the pathogenesis of several cell disorders such as neurodegeneration and cancer. In these settings, pro-oxidant conditions activate many redox-sensitive proteins involved in the regulation of the apoptotic program. Among them, the tumor suppressor p53 and the mitogen-activated protein kinases (MAPK) are implicated both in the pathogenesis of neurodegenerative disorders and in many chemotherapeutic strategies aimed to the elimination of cancer-prone cells from the replicative pool. Recently, AMP-activated protein kinase (AMPK) has been identified as a component of the signaling cascade able to sense bioenergetic and oxidative challenges. Moreover, its capability to trigger apoptosis by activating p53 or some members of the MAPK family (e.g. p38MAPK), makes it a putative player both in neurodegenerative disease and in cancer management. On the basis of this knowledge, this PhD thesis is aimed to elucidate the pro-apoptotic properties of AMPK in SH-SY5Y neuronal cell line under pro-oxidant conditions associated to bioenergetic impairment. This research has been performed by using three compounds known to affect cellular redox homeostas: Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N’]copper(II) perchlorate (Cu(isaepy)2), tetrahydrobiopterin (BH4) and sodium nitroprusside (SNP). Our experiments demonstrated that Cu(isaepy)2, by acting as a DLC-like molecule, was able to induce cell death by activating the AMPK/ p38MAPK/p53 signaling axis, in response to a mitochondrial impairment. The cross-talk between these proteins was found to be operative also in SH-SY5Y cell death induced by BH4, an obligatory cofactor for tyrosine hydroxylase in dopamine synthesis, and to be sensitive to the alteration of glycolytic metabolism. Finally the observation that the iron-nytrosil complex SNP was unable to affect cellular energetics, explains on the one hand the uneffectivenes of AMPK in mediating SNP-induced apoptosis and, on the other, suggests a possible role for this energy-responsive kinase in apoptosis engagement only under condition of bioenergetic stress.

The first section of this dissertation describes the ichthyofaunal community of a mesohaline Chesapeake Bay April--November. Fish assemblages were sampled using two seine gears, a small seine that sampled 352 m 2 and a 914m haul-seine that sampled 144,473 m2. The small seine collected 32 finfish species. The larger gear captured 31 finfish species, including 17 that were not sampled by small gear. Sampled diversity was greater but density estimates were similar to those determined previously by other investigators using flume-net, drop-ring, and otter trawl techniques. Nocturnal abundance of larger fishes far exceeded daylight. Fish abundance and size distribution varied seasonally in both gears. The second section describes the littoral diet composition of striped bass (Morone saxatilis ), bluefish (Pomatomus saltatrix), and weakfish (Cynoscion regalis). These species were the most abundant piscivores sampled in the littoral zone. as in previous Chesapeake Bay diet studies the importance of pelagic prey increased with predator size, however, littoral diets evidenced greater dietary diversity and dependence on crustacean prey items. The third section consists of bioenergetic models that predict piscivore growth based on estimated nocturnal consumption and test energetic consequences of diel thermal refugia. Bluefish and weakfish were predicted to achieve better growth rates than those evidenced by field samples of aged fishes collected from across random Chesapeake habitats. Models predict that striped bass experience declining condition during warm water periods due to physiological stresses and poor prey acquisition. Theoretical diel relocation models illustrated how important species and age specific metabolic restrictions due to physical parameters are to fish health. Findings suggest that habitat specific physical dynamics may be as important as prey provision in determination of and spatial assessments of essential fish habitats.

Through the 137Cs mass balance method, annual consumption rates were estimated for juvenile Atlantic salmon parr and precocious males, as well as brook trout from 4 sites within the Ste Marguerite river system, Quebec. With explicit age analysis, feeding rates and growth rates were derived on an individual fish and age class basis. These represent the first consumption estimates for Atlantic salmon in the wild. The individual fish approach provided a range of data for a single site, as opposed to a single estimate per age class, allowing for an evaluation of the relationship between consumption and growth for each species or life-history variant. Subsequently, the concept of field maintenance ration was introduced as the intercept of consumption over growth.
Salmonid feeding rates were coupled with density estimates to derive total fish exploitation rates for two streams. The application of age- and site-specific feeding rates derived from the 137Cs mass balance method, solved a long standing paradox in stream ecology as all previously inferred salmonid exploitation rates have been in excess of prey turnover. (Abstract shortened by UMI.)

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative fatale de l'âge adulte, caractérisée par une perte de motoneurones, conduisant à une atrophie et une faiblesse musculaires. Des mutations de la superoxyde dismutase-1 (SOD1) provoquent une forme génétique de SLA. Comme chez les patients atteints de SLA, le modèle animal de SLA, SOD1 mutant, révèle que tous les motoneurones sont inégalement sensibles à l'évolution de la maladie. Les mitochondries, centrales énergétiques des cellules, sont des organelles précocement touchées dans la pathologie de la SLA. Un mécanisme attrayant qui sous-tend la susceptibilité différentielle est la nécessité bioénergétique variable de sous-ensembles distincts de motoneurones. Cela implique que dans le système nerveux central, la demande bioénergétique pourrait moduler le seuil pathologique. Même en l'absence de perte bioénergétique, on peut imaginer une situation dans laquelle une contrainte pathologique modifie le niveau à partir duquel la production ou la livraison de l'ATP devient insuffisant, précipitant la chute des neurones les plus vulnérables. Dans les neurones, la majorité de l'ATP est produite par les mitochondries et l'homéostasie des gradients d'ions est le procédé le plus énergivore. La fonction mitochondriale est moindre pour modifier les propriétés électriques des motoneurones si la disponibilité en ATP devient insuffisante pour permettre aux pompes ioniques de maintenir des gradients appropriés. Nous avons démontré que la concentration intracellulaire basale d’ATP dans des cultures de neurones moteurs est diminuée dans les cellules mutées SOD1 par rapport au type sauvage. Paradoxalement à ce résultat, le taux de consommation d'oxygène des mitochondries est augmenté dans les motoneurones SOD1m et il n'existe aucune preuve d'une augmentation de la consommation. Nos résultats appuient l'hypothèse intéressante qu'il y a un découplage entre la chaîne respiratoire et la production d'ATP. Ce découplage peut être utilisé comme une stratégie pour minimiser les propriétés toxiques des mitochondries hyper stimulées
Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder characterized by a loss of motor neurons, leading to muscle wasting and weakness. Mutations in superoxide dismutase-1 (SOD1) cause a form of ALS. As in ALS patients, the mutant SOD1 animal model of ALS reveals that not all motor neurons are equally susceptible to the disease process. An attractive mechanism underlying differential susceptibility is the variable bioenergetics need of distinct subsets of motor neurons. This implies that within the CNS, bioenergetics can modulate the pathological threshold. Even in the absence of loss in bioenergetics, one can envision a situation in which a pathological stress alters the level at which either the production or delivery of ATP becomes insufficient, precipitating the demise of the most vulnerable neuron types. In neurons, majority of ATP is produced by mitochondria and the homeostasis of ion gradients is the most energy-consuming process. Reduced mitochondrial function will modify the electrical properties of motor neurons if ATP availability becomes insufficient to allow ion pumps to maintain appropriate gradients. We demonstrated that the basal ATP intra-cellular concentration in motor neuron cultures lower in SOD1 mutated cells compared to wild type. Paradoxically to this result, the oxygen consumption rate of mitochondria is increase in mSOD1 cells and there is no evidence for an increase of consumption. Our results support the interesting hypothesis that there is an uncoupling between the respiratory chain and the ATP production. This uncoupling might be used as a strategy to minor the toxic properties of hyper stimulated mitochondrion

During tumorigenesis many oncogenic pathways are reprogrammed and modulate mitochondrial metabolism, decreasing oxidative phosphorylation (OXPHOS) activity. They inhibit ATP synthesis and ROS production from OXPHOS complexes favoring metabolic switching toward a more glycolytic metabolism and tumor growth. This metabolic rewiring is well established in solid tumor but very little is known about this event in non-solid tumors such as leukemias. Indeed, it has been demonstrated that in chronic lymphocytic leukemia (B-CLL), cells show a high OXPHOS activity and elevated ROS levels. Notably, expression of the p66Shc protein, whose mitochondrial fraction modulates cell bioenergetics and increases ROS generation, is lost during B-CLL tumorigenesis. I investigate the effects of the expression of p66Shc on bioenergetics and viability of B-CLL cells. After p66Shc expression in MEC-1 cells, a B-CLL cell model, a fraction of it is in the intermembrane space of mitochondria. p66Shc expression decreases both basal and maximal respiration, decreases mitochondrial membrane potential, total ATP levels and renders cells less dependent from OXPHOS to ATP supply without changes mitochondrial mass. I propose that decreased mitochondrial respiration is due to decreased activity of respiratory complex I and II. In more detail, there is a decrease in complex I assembly with a consequent inhibition of its activity, and a decrease in complex II activity caused by a multi-protein complex in which TRAP1 and active ERK are involved. p66Shc expression in MEC-1 cells increases mitochondrial ERK activation. The mitochondrial fraction of ERK contribute to tumor cell survival by inhibiting opening of the permeability transition pore (PTP), a mitochondrial channel whose opening irreversibly commits cells to death. I check the effect of p66Shc expression on cell viability. Although its pro-apoptotic function well described in many cell models, in MEC-1 cells p66shc protects cells from death induced by mitochondrial-derived oxidative stress derived from starvation, EM20-25 and CisPlatin treatments. In depletion conditions, p66Shc expression correlates with more active mitochondrial ERK and the inhibition of ERK decreases cell viability. Inhibition of mitochondrial chaperones activity with Cyclosporine A, an inhibitor of CyP-D, or 17AAG, an inhibitor of TRAP1, also affects cell viability under depletion condition, thus supporting the hypothesis that mitochondrial ERK is involved in survival mechanism induced by p66Shc expression. Taken together these data indicate that regulation of RC complex activity can be linked to regulation of cell survival. We hypothesize that the mitochondrial branch of the Ras-ERK signaling pathway regulates mitochondrial bioenergetics by affecting SDH activity and resistance to apoptosis of B-CLL cells. Through the activation of mitochondrial ERK and inhibition of SDH activity, p66Shc can decrease OXPHOS activity and ROS production, a condition that is necessary in the early stages of tumorigenesis. It can also support tumour cell growth making mPTP less sensitive to opening by activating mitochondrial ERK.
Durante lo sviluppo tumorale, molte vie di segnale delle cellule vengono riprogrammate cambiando il metabolismo mitocondriale e diminuendo l’attività della fosforilazione ossidativa. In questa maniera viene diminuita la produzione di ATP e di ROS da parte della fosforilazione ossidativa favorendo un metabolismo glicolitico e la crescita tumorale. I meccanismi alla base dello sviluppo di questo fenotipo sono ben caratterizzati nelle cellule di tumori solidi, ma poco si sa a riguardo delle leucemie. Nella leucemia linfocitica cronica, le cellule presentano elevata attività OXPHOS e alti livelli di ROS. È noto che la proteina p66Shc, di cui una frazione si trova nello spazio intermembrana del mitocondrio, modula la bioenergetica cellulare e induce la produzione di ROS. Durante il processo di tumorigenesi della B-CLL l’espressione di p66Shc viene persa. Quindi, sono andata ad analizzare l’effetto della espressione di p66Shc sulla bioenergetica e la vitalità cellulare della leucemia linfocitica cronica. Dopo l’espressione di p66Shc nel modello cellulare di leucemia linfocitica cronica di tipo B, MEC-1, una frazione della proteina è stata trovata nello spazio intermembrana dei mitocondri. L’espressione di p66Shc nelle cellule diminuisce la respirazione mitocondriale, massima e basale, ma anche il potenziale di membrana e i livelli totali di ATP. Ho proposto che queste differenze sono dovute alla diminuzione dell’attività dei complessi respiratori: complesso I e complesso II indotta dalla espressione di p66Shc nelle cellule MEC-1. In particolare, il complesso I è meno assemblato con la conseguente diminuzione della sua attività mentre il complesso II diminuisce la sua attività a causa di un complesso multi proteico in cui sono coinvolti la proteina chinasi ERK e lo sciaperone mitocondriale TRAP1. L’espressione di p66Shc nelle MEC-1 aumenta l’attivazione della frazione mitocondriale di ERK. Questa frazione, contribuisce alla sopravvivenza delle cellule tumorali inibendo l’apertura del poro di transizione di permeabilità (mPTP), un canale mitocondriale la cui apertura porta alla morte cellulare. Ho quindi analizzato l’effetto dell’espressione di p66Shc sulla vitalità cellulare delle MEC-1. Sebbene, p66Shc ha una attività pro-apoptotica, ho osservato che la sua espressione nelle MEC-1 protegge le cellule dalla morte cellulare indotta dallo stress ossidativo prodotto dal mitocondrio da trattamenti come EM20-25, CisPlatino e assenza di glucosio. In assenza di glucosio, l’espressione di p66Shc correla con una maggiore attivazione di ERK nel mitocondrio. L’inibizione di ERK diminuisce la vitalità cellulare. In assenza di glucosio, anche l’inibizione degli sciaperoni mitocondriali CyP-D e TRAP1 influenzano la vitalità cellulare. Quindi, la frazione mitocondriale di ERK è coinvolta nella regolazione delle vie di segnale che proteggono le cellule dalla morte cellulare dopo l’espressione di p66SHhc. Questi dati, indicano che la regolazione dell’attività dei complessi respiratori è legata alla regolazione della sopravvivenza cellulare. Abbiamo ipotizzato che nella leucemia linfocitica cronica la parte mitocondriale della via di segnale RAS-ERK può regolare la bioenergetica influenzando l’attività enzimatica del complesso II e la resistenza alla morte cellulare. Inoltre, abbiamo ipotizzato anche una nuova funzione per p66Shc in cui, attraverso l’attivazione di ERK e l’inibizione dell’attività del complesso II può diminuire l’attività della fosforilazione ossidativa e i livelli di ROS, una condizione necessaria nelle prime fasi della tumori genesi. p66Shc può anche sostenere la crescita tumorale rendendo mPTP meno sensibile all’apertura attraverso l’attivazione della frazione mitochondrial di ERK.

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This study has the objective to analyze the relationship between body and mind in Alexander Lowen´s publications to discuss how the body illness is related to the character structures. For this, we seek to know how the body appears as a concept in the perspective presented by the author; discuss how is and how historically builds the relationship between body and mind in his approach; know how the character and illness concepts were built in his theory; discuss how the author develops the relationship between both. It was assumed as hypothesis that the character appears as an important joint point between mind and body and that its constitution is related to the body illness. Bibliographic and qualitative research was used, and the method used was Categorical Content Analysis of Lowen publications, his precursors and also of current articles on the topic. Five categories were developed to analyze the collected information: child development; the concept of energy; the relationship between the ego and the body; the emotions and sexuality; and the individual's relationship with the historical and social context in which they live. We conclude that the character appears as a fundamental and resulting element in the relationship between mind and body and that its constitution follows the same function of the illness, which is to react to a stressor element and restore the body's balance. Therefore, there is a relationship between them, since different types of character handle the particular disease forms. It is noticed that individuals with a combination of oral and rigid traits have unique characteristics that make them more prone to bodily illness than individuals with other character structures. This way of categorization changes among the author´s publications. We emphasize the importance of the concepts discussed by Lowen in structuring his theory and in developing a perspective in psychology that addresses the body and mind in an integrated manner.
O presente trabalho tem como objetivo analisar a relação entre corpo e mente na obra de Alexander Lowen, no intento de discutir como o adoecimento corporal se relaciona com as estruturas de caráter. Para isso, busca-se: a) conhecer como o corpo aparece como conceito na perspectiva apresentada pelo autor; b) analisar como se dá e como se constrói historicamente a relação entre corpo e mente nessa abordagem; c) conhecer como se constroem e se fundamentam os conceitos de caráter e adoecimento em sua teoria; d) debater como o autor desenvolve a articulação entre ambos. Assumiu-se como hipóteses que o caráter se mostra como um ponto de articulação importante entre mente e corpo e que sua constituição está relacionada ao adoecimento corporal. Foi utilizada pesquisa bibliográfica, qualitativa, e o método utilizado foi a Análise de Conteúdo Categorial das obras de Lowen, assim como de seus precursores, bem como artigos atuais sobre a temática. Cinco categorias foram elaboradas para a análise dos dados coletados: o desenvolvimento infantil; o conceito de energia; a relação entre o ego e o corpo; as emoções e a sexualidade; e a relação do indivíduo com o contexto sócio-histórico em que vive. Conclui-se que o caráter se mostra como elemento fundamental e resultante na relação entre corpo e mente. Além disso, sua constituição segue a mesma função do adoecimento, que é a de reagir a um agente estressante e restabelecer o equilíbrio do organismo. Portanto, há uma relação entre ambos, uma vez que diferentes tipos de caráter lidam com a doença de formas particulares. Percebe-se que indivíduos com a combinação de traços orais e rígidos possuem características peculiares que os tornam mais propensos ao adoecimento corporal do que indivíduos com outras estruturas de caráter, embora essa forma de categorização se modifique ao longo da obra do autor. Ressalta-se a importância dos conceitos discutidos por Lowen na estruturação de sua teoria e no desenvolvimento de uma perspectiva em psicologia que aborde o corpo e a mente de forma integrada.

Primary CoQ10 deficiency diseases encompass a heterogeneous spectrum of clinical phenotypes. Among these, defect or mutation on COQ2 gene, encoding a para-hydroxybenzoate polyprenyl transferase, have been associated with different diseases. Understanding the functional and metabolic impact of COQ2 mutation and the consequent CoQ10 deficiency is still a matter of debate. To date the aetiology of the neurological phenotypes correlated to CoQ10 deficiency does not present a clear genotype-phenotype association. In addition to the metabolic alterations due to Coenzyme Q depletion, the impairment of mitochondrial function, associated with the reduced CoQ level, could play a significant role in the metabolic flexibility of cancer. This study aimed to characterize the effect of varying degrees of CoQ10 deficiency and investigate the multifaceted aspect of CoQ10 depletion and its impact on cell metabolism. To induced CoQ10 depletion, different cell models were used, employing a chemical and genome editing approach. In T67 and MCF-7 CoQ10 depletion was achieved by a competitive inhibitor of the enzyme, 4-nitrobenzoate (4-NB), whereas in SH-SY5Y the COQ2 gene was edited via CRISPR-Cas9 cutting edge technology.

O fungo dimórfico Paracoccidioides brasiliensis, é o agente etiológico da paracoccidioidomicose, uma das micoses sistêmicas humanas mais prevalentes na América Latina. Componentes da cadeia respiratória mitocondrial são potenciais alvos quimioterapêuticos. Nesse sentido, em nosso trabalho demonstramos diferenças entre a cadeia respiratória do fungo P. brasiliensis e do hospedeiro mamífero. A respiração, potencial de membrana e fosforilação oxidativa mitocondrial de esferoplastos de P. brasiliensis foram avaliados in situ, os quais demonstraram a presença de uma cadeia respiratória funcional. A adição de ADP induziu a transição da respiração do estado de repouso para o fosforilativo, em esferoplastos energizados com succinato, NADH, NAD+ e substratos ligados ao complex I. A presença de uma NADH-ubiquinona oxidoredutase foi demonstrada pela capacidade das células oxidarem NADH exógeno, assim como, pela respiração insensível a rotenona e sensível a flavona. Além disso, a respiração mantida por NAD+ foi sensível a rotenona e flavona, sugerindo que vias metabólicas citosolicas contribuem para a produção de NADH e substratos ligados ao complexo I. Foi demonstrado também que os níveis de expressão do complexo I e da NADH desidrogenase alternativa alternam-se durante a curva de crescimento de leveduras de P. brasiliensis. A respiração induzida por NADH ou succinato foi parcialmente inibida por KCN ou antimicina A e sensível à inibição por BHAM, indicando a presença de uma oxidase alternativa. A fim de se caracterizar esta enzima oxidase alternativa, o seu gene foi clonado e expressado em xii S. cerevisiae e E. coli, o qual conferiu uma respiração resistente a cianeto e sensível a BHAM. S. cerevisiae expressando oxidase alternativa mostraram uma menor taxa de multiplicação celular e diminuição na geração de EROs. Nós também observamos que inibidores da cadeia transportadora de elétrons retardaram a transição de micélio para levedura em culturas de P. brasiliensis. Além disso, estes inibidores e outras drogas geradoras de EROs aumentaram a expressão do gene da oxidase alternativa, assim como a produção de EROs em leveduras de P. brasiliensis.
Paracoccidioides brasiliensis, a thermically dimorphic fungus, is the etiological agent of endemic paracoccidioidomycosis, one of the most prevalent human systemic mycosis in Latin America. Components of the respiratory chain constitute potential pharmacological targets, and here are reported differences between the respiratory chain of the mammalian host and the fungus P. brasiliensis. Respiration, membrane potential and oxidative phosphorylation of mitochondria from P. brasiliensis spheroplasts were evaluated in situ, which demonstrated the existence of a functional respiratory chain. Adenosine 5\'-diphosphate (ADP) induced a transition from resting to phosphorylating respiration in mitochondria energized by succinate, NADH, NAD+ and complex I linked substrates. The presence of an alternative NADH-ubiquinone oxidoreductase was indicated by: the ability of the fungus to oxidize exogenous NADH; the insensitivity substrate-supported respiration to rotenone and sensitivity of this respiration to flavone. In addition, the sensitivity of NAD+-supported respiration to rotenone and flavone suggest that citosolic pathways contribute to NADH and complex I linked substrates production. Moreover, it was demonstrated that expression levels of complex I and alternative NADH dehydrogenase change during P. brasiliensis growth curve. The partial sensitivity of NADH or succinate-supported respiration to antimycin A and cyanide, as well as the sensitivity to BHAM, indicates the presence of an alternative oxidase. Therefore, to characterize the alternative oxidase its gene was cloned and heterologously xii expressed in S. cerevisiae and E. coli, wich confered, a cyanide resistant respiration and BHAM sensitivity. Moreover, S. cerevisiae that expressed alternative oxidase showed slower growth rate and decreased ROS generation. We also observed that electron transport pathways inhibitors delayed the P. brasiliensis mycelium to yeast transition in cultures. Besides, these inhibitors and other ROS generated drugs increase the ROS production and altenative oxidase expression.

BACKGROUND Maternal obesity (MO) is expanding worldwide, contributing to Gestational Diabetes Mellitus (GDM) prevalence. The altered placental function and intrauterine environment of MO and GDM are associated with adverse maternal and fetal outcomes, with short- and long-term complications. Growing evidence suggests that both MO and GDM are characterized by placental autophagy disturbances, mitochondrial (mt) dysfunctions and epigenetic alterations, which could contribute to the pathogenesis of pregnancy-related metabolic diseases. For these reasons the aim of this work was to characterize placental autophagy and mitochondrial bioenergetics, and maternal plasmatic microRNAs profile in the context of MO and GDM. METHODS 60 women with spontaneous singleton pregnancies delivering by elective Caesarean section were enrolled: 25 normal-weight (NW), 22 obese without comorbidities (OB/GDM(-)), 13 obese with GDM (OB/GDM(+)). The expression of antioxidant metabolism markers and autophagy-related genes expression was evaluated in placental villous tissue by Real-time PCR with SYBR green chemistry. In placenta, we also quantified ATP by CellTiter-Glo Luminescent Cell Viability Assay adapted to placental villous tissue, and mt Complexes I-V protein content by Western Blot experiments. Moreover, in a subgroup of 7 NW, 6 OB/GDM(-) and 6 OB/GDM(+), microRNA (miRNA) profiling in maternal plasma at delivery was performed with miRCURY LNA Serum/Plasma miRNA Focus PCR Panel (Qiagen). Statistical analysis and bioinformatics predictive tools: SPSS v.27 (IBM); GeneGlobe (Qiagen); miTALOS v.2; miRPath v.3. RESULTS AND CONCLUSIONS Placental antioxidant genes expression (CAT, GPX1, GSS, GSR) tended to decrease, the pro-autophagic ULK1 increased and the chaperone-mediated autophagy (CMA) regulator PHLPP1 decreased in OB/GDM(-) vs NW. On the other hand, PHLPP1 expression increased in OB/GDM(+) vs OB/GDM(-). These preliminary findings showed placental alterations of autophagy in MO and GDM, paving the way for further analyses aimed at elucidating the role of placental autophagy in metabolic disorders during pregnancy. Moreover, placentas of OB/GDM(-) and OB/GDM(+) women had reduced ATP level vs NW. All OB showed decreased Complex I vs NW. Furthermore, lower expression of Complexes III and V was found in OB/GDM(+) group (vs NW; vs NW and OB/GDM(-), respectively), along with a reduced fetal/placental weight ratio (i.e. placental efficiency) vs NW. These results might suggest impaired placental mt function in obese subjects, especially with GDM. Sexual dimorphism in terms of the above-mentioned placental molecular markers was also investigated, with intriguing results suggesting that females and males might respond differently to insults and/or have differential, sex-dependent compensatory mechanisms in order to deal with the perturbed environment. MiRNA profiling in maternal plasma highlighted patterns of significantly differentially expressed miRNAs between the three groups. In particular, OB/GDM(-) vs NW differed in the expression of 4 miRNAs, OB/GDM(+) vs NW of 1, and OB/GDM(+) vs OB/GDM(-) of 14. Pathway enrichment analysis by miRPath and miTALOS revealed many pathways associated with the set of miRNAs of each comparison, among which the most interesting and relevant in our context were related to nutrients and hormones metabolism, inflammation and oxidative stress. Indeed, miRNAs could be promising biomarkers of metabolic alterations in MO and GDM. Nevertheless, future investigations are needed to further deepen the pregnancy epigenetic landscape.

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This work aims to verify that the contributions of verbal and bodily therapeutic interventions of Bioenergetic Analysis in the treatment of patients with primary and secondary complaints of organic pain. This intention is consistent with international efforts in researching the therapeutic efficacy of body psychotherapy. We start from the hypothesis that the diagnosis and intervention in the body with neo-Reichian techniques can serve as catalysts in the process of patient care traditionally regarded as psychosomatic. We focused on a qualitative method to clinical research, in which eight patients with various somatic complaints were submitted to twelve sessions of psychotherapy in bioenergetics and evaluated by instruments measuring psychological stress and general health conditions before and after intervention therapy. Data are presented through a characterization of the social profile of the patients, as well as through case studies, linking with the results obtained in the tests. Some issues raised by patients confirm the working hypotheses discussed in the literature, such as development history of trauma and family violence, abandonment experiences, coldness and detachment from the mother figure and a bond ambivalent love / heartbreak with the father figure; difficulties to experience pleasure in life and sexuality; emerging symptom associated with a particular crisis of life, due to a chronic disease process. Tests have shown mixed results, which are discussed in accordance with the circumstances in each case as indicative apparent adverse findings show that obtained in the process of intervention can increase the psychic stress, resulting in a crisis condition required for significant changes. Despite this, patients report improvements in relation to the disease and how to regulate their emotions, emphasizing the importance of bioenergetic exercises in the care and procedures. We conclude with the belief that a specific listening to body in the therapeutic process serves as a communication channel that facilitates empathy in the integrated health and the need to broaden the scope of this research, with additional experimental procedures
Esse trabalho busca verificar quais as contribuições terapêuticas das intervenções verbais e corporais da Análise Bioenergética no tratamento de pacientes com queixas primárias e secundárias de sofrimento orgânico, coadunando com os esforços internacionais em pesquisar a eficácia da psicoterapia corporal. Partimos da hipótese que o diagnóstico e intervenção corporais na vertente neorreichiana podem servir como catalisadores no processo de cuidado de pacientes tradicionalmente considerados como psicossomáticos. Privilegiou-se um método qualitativo clínico de pesquisa, no qual oito pacientes, com queixas somáticas variadas, foram submetidas a doze sessões de psicoterapia bioenergética, sendo avaliadas por instrumentos psicológicos de medição do estresse e de condições de saúde geral, antes e depois da intervenção. Os dados são apresentados através de uma caracterização do perfil social das pacientes, bem como através de estudos de caso, articulando com os resultados obtidos nos testes. Alguns temas levantados pelas pacientes confirmam hipóteses de trabalho discutidas na literatura, tais como: histórico de traumas de desenvolvimento e violência intrafamiliar; experiências de abandono, frieza e distanciamento da figura materna e um vínculo ambivalente de amor/mágoa com a figura paterna; dificuldades de experimentar prazer na vida e na sexualidade; o sintoma surgindo associado a uma crise específica de vida, decorrente de um processo de adoecimento crônico. Os testes demonstraram resultados variados, que são discutidos de acordo com o contexto de cada caso, pois aparentes indicativos desfavoráveis revelam que as descobertas obtidas no processo de intervenção podem aumentar a tensão psíquica, resultando num estado de crise necessária para mudanças importantes. Apesar disso, as pacientes relatam melhoras na relação com a doença e na forma de regular suas emoções, enfatizando a importância dos exercícios e procedimentos nos atendimentos. Concluímos o trabalho com a convicção de que a escuta diferenciada do corpo no processo terapêutico serve como canal de comunicação que facilita a empatia no cuidado integrado em saúde, bem como a necessidade de ampliar o escopo dessa pesquisa, com procedimentos experimentais complementares

Primena biomarkera ćelijske energetike, uključujući indikatore metabolizma kreatina u krvi, je relativno novijeg datuma, gde se ovi indikatori koriste kao mogući pokazatelji stanja organizma pri maksimalno intenzivnim fizičkim aktivnostima. Cilj istraživanja je obuhvatao utvrđivanje efekata pojedinačnih epizoda aerobnog i anaerobnog vežbanja maksimalnog intenziteta na biomarkere perifernog zamora i ćelijske bioenergetike kod mladih muškaraca i žena. Istraživanje je dizajnirano tako da obuhvati populaciju fizički aktivnih muškaraca i žena, kao i populaciju aktivnih sportista. U prvom eksperimentalnom tretmanu fizički aktivni ispitanici muškog (n =12) i ženskog pola (n = 11) podvrgnuti su test protokolima aerobnog i anaerobnog opterećenja maksimalnog intenzivnog i kratkog trajanja. Tokom aerobnog test protokola ispitanici su trčali do maksimalnog voljnog otkaza na tredmil traci sa progresivnim povećanjem opterećenja. Pri anaerobnom test protokolu ispitanici su izvršili testiranje snažne izdržljivosti gornjih ekstremiteta do otkaza potiskom sa ravne klupe, uz opterećenje od 25% od njihove telesne težine. Drugi eksperimentalni tretman je sačinjen iz pre-eksperimentalnog testiranja kardiorespiratorne forme i eksperimentalne protokol sesije trčanja do maksimalnog voljnog otkaza na pokretnoj traci, pri konstantnoj individualnoj brzina trčanja na anaerobnom pragu. U ovom eksperimentalnom tretmanu bila je uključena populacija aktivnih sportista     (n = 10). Pre, tokom i nakon eksperimentalnih sesija praćena je koncentracija različitih biohemijskih i hematoloških markera: guanidinosirćetna kiselina (GAA); kreatin (Cr); kreatinin (Crn); laktat (Lac); interleukin-6 (IL-6); kreatin kinaza (CK); kortizol (Cor). Rezultati prvog eksperimentalnog tretmana su utvrdili statistički značajne promene u koncentraciji GAA, Cr i Crn u ktvi pre i nakon pojedinačne epizode aerobnog i anaerobnog vežbanja maksimalnim intenzitetom. Utvrđena je i statistički značajna povezanost između vežbanjem-indukovanih promena u cirkulatornim vrednostima GAA, Cr, Crn za vreme pre, tokom i nakon drugog eksperimentalnog tretmana. Uočena je statistički značajna povezanost između promena koncentracije GAA, Cr, Crn u serumu sa tradicionalnim biomarkerima perifernog zamora (IL6, Cor, Lac, CK). Dobijeni rezultati ukazuju na mogućnost primene biomarkera metabolizma kreatina u krvi prilikom praćenja i evaluacije stanja organizma tokom maksimalnih intenzivnih fizičkih aktivnosti kod mladih muškaraca i žena.
The use of biomarkers of cellular bioenergetics in exercise science appears more prevalent in recent years, where these outcomes perhaps describe changes in creatine metabolism during strenuous exercise. The aim of this study was to determine the effects of individual episodes of strenuous aerobic and anaerobic exercise on several biomarkers of peripheral fatigue and cellular bioenergetics in young men and women. The study recruited physically active men and women, and active athletes. In the first experiment, physically active men (n = 12) and women (n = 11) were subjected to strenuous aerobic and anaerobic exercise. During the aerobic test, subjects ran to exhaustion while during the anaerobic test, subjects performed repetitive bench press exercise. The second experimental treatment consisted of a pre-experimental testing of cardiorespiratory fitness, and an experimental protocol of a strenuous running session to exhaustion at constant individual running speed at the anaerobic threshold; active athletes (n = 10) were included in this experimental treatment. The blood levels of various biochemical and hematological markers were monitored before, during and after the experimental sessions, including guanidinoacetic acid (GAA); creatine (Cr); creatinine (Crn); lactate (Lac); interleukin-6 (IL-6); creatine kinase (CK); cortisol (Cor), and plethora of other physiological outcomes. We found statistically significant changes in serum GAA, Cr and Crn before and after a single session of strenuous aerobic and anaerobic exercise. A significant correlation was found between exercise-induced changes in serum GAA, Cr and Crn before, during and after the second experimental intervention. A statistically significant association was observed between changes in serum GAA, Cr, Crn and traditional biomarkers of peripheral fatigue (IL6, Cor, Lac, CK). The results of the present study suggest that biomarkers of creatine metabolism might be used as innovative tools in monitoring strenuous exercise in young men and women.

Chez les mâles, la testostérone joue un rôle important dans l'expression des signaux sexuels attractifs pour la femelle. Cependant, de nombreuses études mettent en évidence l'effet négatif de la testostérone sur les défenses immunitaires ou la résistance aux parasites. Selon l'hypothèse du handicap immunitaire (ICHH, Folstad et Karter, 1992), ce compromis permettrait d'assurer l'honnêteté du signal sexuel ; les mauvais mâles ne pouvant pas exprimer de signaux sexuels de bonne qualité tout en gérant l'immunosuppression due à la testostérone. Ma thèse a pour objectif de définir le rôle de la testostérone dans l'honnêteté des signaux sexuels dans un contexte de communication multimodale. Elle permet également de comprendre les mécanismes physiologiques par lesquels la testostérone module les signaux sexuels acoustiques et visuels chez les mâles Hyla arborea, un amphibien connu pour utiliser ces deux types de signaux pour attirer les femelles. Nos résultats montrent que les signaux sexuels acoustiques et visuels chez cette espèce sont testostéronedépendants. En revanche, puisque la testostérone n'a pas d'effet immunosuppresseur dans notre étude, elle ne serait pas le médiateur direct de l'honnêteté des signaux chez cette espèce. D'un point de vue physiologique, l'effet de la testostérone sur le chant s'explique par l'amélioration des propriétés contractiles des muscles du tronc responsables du chant lié à une augmentation de l'efficacité mitochondriale. D'autre part, les analyses biochimiques montrent que la couleur du sac vocal varie linéairement avec la quantité de pigments caroténoïdes présents dans le plasma, qui pourrait être testostérone-dépendant. Enfin, cette thèse montre également que la testostérone agit sur le comportement de chasse permettant l'acquisition de ressources énergétiques et de caroténoïdes pour le chant et la couleur des mâles
In males, testosterone plays an important role on attractive sexual signal expression. However, numerous studies highlighted that testosterone also has negative effects on immunity defenses and parasite resistance capacity. According to the immunocompetence handicap hypothesis (ICHH, Folstad and Karter, 1 992), this trade-off assures signal honesty because only good males could express powerful signals while managing the immunosuppression of testosterone. My thesis aims to define the role of testosterone on the signal honesty in a multimodal communication context. Moreover it allows us to understand some physiological mechanisms involved in the modulation of acoustic and visual signals in males Hyla arborea, an amphibian known to use both acoustic and visual signals to attract females. Our results show that acoustic and visual signals are testosterone-dependent. In contrast, since testosterone has no immunosuppression effect in our study, it would not be the direct mediator of the signal honesty in this species. From a physiological point of view, the testosterone effects on calls could be explained through the positive effect of testosterone on trunk muscle mitochondrial efficacy and their contractile properties. Besides, biochemical analyses show that vocal sac coloration varies with the amount of carotenoid pigments present in the plasma that could be testosterone-dependent. Finally, this thesis also assumes that testosterone acts on the hunting behavior permitting the acquisition of energy resources and carotenoids for singing and coloration of males

Tamarindus indica L. é um componente natural amplamente consumido por humanos, apresentando propriedade antiinflamatória, antidiabética e antihepatotóxica. Além disso, como nós demonstramos previamente, o extrato apresenta atividade hipolipêmica e antioxidante. Neste trabalho, nós demonstramos o efeito do extrato de T. indica sobre mitocôndrias isoladas de fígado de rato. Na presença de Ca2+, o extrato causou um inchamento osmótico mitocondrial concentração-dependente, associado ao aumento da velocidade basal (V4), dissipação do potencial de membrana e liberação do Ca2+ pré-acumulado, os quais foram inibidos por ciclosporina A (CsA) e assim atribuídos a transição de permeabilidade da membrana mitocondrial (TPMM). A indução do inchamento osmótico mitocondrial foi prevenida por EGTA e vermelho de rutênio, indicando a dependência da TPMM de Ca2+. A oxidação de proteínas tiol mitocondrial, um mecanismo bem estabelecido como causador da TPMM, foi detectado. Entretanto, nenhuma alteração significante foi observada no estado redox de GSH. A oxidação de NAD(P)H e o acúmulo de espécies reativas de oxigênio produzidas pela mitocôndria foram observadas, em ambos os casos, foram prevenidas por CsA e/ou EGTA, indicando que são conseqüências da TPMM induzida pelo extrato de T. indica. Portanto, sem aparente envolvimento do estresse oxidativo no processo, sugerindo uma interação direta de compostos do extrato com grupos tiólicos de proteínas da membrana. O extrato de T. indica induziu uma depleção de ATP associada à TPMM, mostrando assim potencial para causar a morte celular por apoptose ou necrose, resultante da indução da TPMM por si ou pela depleção do ATP via TPMM. In vitro, o extrato apresentou capacidade scavenger de radicais livres, verificado pelo ensaio do DPPH, radical superóxido e radical hidroxil; e ainda foi capaz de diminuir a lipoperoxidação mitocondrial. Além disso, em baixas concentrações, o extrato mostrou uma propriedade quelante de Fe2+.
Tamarindus indica L. is a natural dietary component widely consumed by humans, presenting well established anti-inflammatory, anti-diabetic and anti-hepatotoxic properties. In addition, as we have previously demonstrated, extract presents hypolipemic and antioxidant activities. We show here the effects of T. indica extract on isolated rat liver mitochondria. In the presence of Ca2+, the extract caused mitochondrial concentration-dependent swelling, associated to, resting respiration increase (V4), membrane potential dissipation and release of pre-accumulated Ca2+, inhibited by cyclosporine A (CsA) and thus ascribable to mitochondrial permeability transition (MPT). This swelling induction was prevented by EGTA and ruthenium red, indicating strict dependence of MPT on Ca2+. Oxidation of mitochondrial membrane protein thiols, a well established mechanism causing MPT was detected. However, no significant change was observed in the GSH redox state, and the NADPH oxidation and accumulation of mitochondria-generated reactive oxygen species that was observed, were prevented by CsA and/or EGTA, indicating that they are consequence of the MPT induced by T. indica extract. Therefore, no apparent oxidative stress condition is involved as cause of this process suggesting that direct interaction with membrane protein thiol groups of the compounds responsible for MPT induction occurs. T. indica extract led to MPTassociated ATP depletion, thus showing the potential to cause cell death by apoptosis or necrosis resulting from MPT induction per se or from ATP depletion by MPT. In vitro, the extract presented free radical scavenging ability, as assessed by the 2,2-diphenyl-1- picrylhydrazyl (DPPH), superoxide radicals and radical hydroxyl assays, and led to decreased lipid peroxidation in mitochondria, as assessed by the thiobarbituric acid reactive substances (TBARS) assay. In addition, the extract showed an iron chelanting property in low concentrations.