Modeling Ultrafast Spectroscopy of the NADH Dimer: From UV-VIS to X-rays

Ultrafast pump-probe spectroscopy is a conceptually simple and versatile tool for resolving photoinduced dynamics in molecular systems. Due to the fast development of new experimental setups, such as synchrotron light sources and X-ray free electron lasers (XFEL), new spectral windows are becoming accessible. On the one hand, these sources have enabled scientist to access faster and faster time scales and to reach unprecedent insights into dynamical properties of matter. On the other hand, the complementarity of well-developed and novel techniques allows to study the same physical process from different points of views, integrating the advantages and overcoming the limitations of each approach. In this context, it is highly desirable to reach a clear understanding of which type of spectroscopy is more suited to capture a certain facade of a given photo-induced process, that is, to establish a correlation between the process to be unraveled and the technique to be used. In this thesis, I will show how computational spectroscopy can be a tool to establish such a correlation. I will study a specific process, which is the ultrafast energy transfer in the nicotinamide adenine dinucleotide dimer (NADH). This process will be observed in different spectral windows (from UV-VIS to X-rays), accessing the ability of different spectroscopic techniques to unravel the system evolution by means of state-of-the-art theoretical models and methodologies. The comparison of different spectroscopic simulations will demonstrate their complementarity, eventually allowing to identify the type of spectroscopy that is best suited to resolve the ultrafast energy transfer.

Fatty Acid Synthase Inhibitors Induce Apoptosis In Non-tumorigenic Melan-a Cells Associated With Inhibition Of Mitochondrial Respiration.

The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition.

Taurine Supplementation Reduces Blood Pressure And Prevents Endothelial Dysfunction And Oxidative Stress In Post-weaning Protein-restricted Rats.

Taurine is a sulfur-containing amino acid that exerts protective effects on vascular function and structure in several models of cardiovascular diseases through its antioxidant and anti-inflammatory properties. Early protein malnutrition reprograms the cardiovascular system and is linked to hypertension in adulthood. This study assessed the effects of taurine supplementation in vascular alterations induced by protein restriction in post-weaning rats. Weaned male Wistar rats were fed normal- (12%, NP) or low-protein (6%, LP) diets for 90 days. Half of the NP and LP rats concomitantly received 2.5% taurine supplementation in the drinking water (NPT and LPT, respectively). LP rats showed elevated systolic, diastolic and mean arterial blood pressure versus NP rats; taurine supplementation partially prevented this increase. There was a reduced relaxation response to acetylcholine in isolated thoracic aortic rings from the LP group that was reversed by superoxide dismutase (SOD) or apocynin incubation. Protein expression of p47phox NADPH oxidase subunit was enhanced, whereas extracellular (EC)-SOD and endothelial nitric oxide synthase phosphorylation at Ser 1177 (p-eNOS) were reduced in aortas from LP rats. Furthermore, ROS production was enhanced while acetylcholine-induced NO release was reduced in aortas from the LP group. Taurine supplementation improved the relaxation response to acetylcholine and eNOS-derived NO production, increased EC-SOD and p-eNOS protein expression, as well as reduced ROS generation and p47phox expression in the aortas from LPT rats. LP rats showed an increased aortic wall/lumen ratio and taurine prevented this remodeling through a reduction in wall media thickness. Our data indicate a protective role of taurine supplementation on the high blood pressure, endothelial dysfunction and vascular remodeling induced by post-weaning protein restriction. The beneficial vascular effect of taurine was associated with restoration of vascular redox homeostasis and improvement of NO bioavailability.

Two-component System Vicrk Regulates Functions Associated With Establishment Of Streptococcus Sanguinis In Biofilms.

Streptococcus sanguinis is a commensal pioneer colonizer of teeth and an opportunistic pathogen of infectious endocarditis. The establishment of S. sanguinis in host sites likely requires dynamic fitting of the cell wall in response to local stimuli. In this study, we investigated the two-component system (TCS) VicRK in S. sanguinis (VicRKSs), which regulates genes of cell wall biogenesis, biofilm formation, and virulence in opportunistic pathogens. A vicK knockout mutant obtained from strain SK36 (SKvic) showed slight reductions in aerobic growth and resistance to oxidative stress but an impaired ability to form biofilms, a phenotype restored in the complemented mutant. The biofilm-defective phenotype was associated with reduced amounts of extracellular DNA during aerobic growth, with reduced production of H2O2, a metabolic product associated with DNA release, and with inhibitory capacity of S. sanguinis competitor species. No changes in autolysis or cell surface hydrophobicity were detected in SKvic. Reverse transcription-quantitative PCR (RT-qPCR), electrophoretic mobility shift assays (EMSA), and promoter sequence analyses revealed that VicR directly regulates genes encoding murein hydrolases (SSA_0094, cwdP, and gbpB) and spxB, which encodes pyruvate oxidase for H2O2 production. Genes previously associated with spxB expression (spxR, ccpA, ackA, and tpK) were not transcriptionally affected in SKvic. RT-qPCR analyses of S. sanguinis biofilm cells further showed upregulation of VicRK targets (spxB, gbpB, and SSA_0094) and other genes for biofilm formation (gtfP and comE) compared to expression in planktonic cells. This study provides evidence that VicRKSs regulates functions crucial for S. sanguinis establishment in biofilms and identifies novel VicRK targets potentially involved in hydrolytic activities of the cell wall required for these functions.

Inactivation Of Ampk Mediates High Phosphate-induced Extracellular Matrix Accumulation Via Nox4/tgfß-1 Signaling In Human Mesangial Cells.

High phosphate (Pi) levels and extracellular matrix (ECM) accumulation are associated with chronic kidney disease progression. However, how high Pi levels contribute to ECM accumulation in mesangial cells is unknown. The present study investigated the role and mechanism of high Pi levels in ECM accumulation in immortalized human mesangial cells (iHMCs). iHMCs were exposed to normal (0.9 mM) or increasing Pi concentrations (2.5 and 5 mM) with or without diferent blockers or activators. NOX4, phosphorylated AMPK (p-AMPK), phosphorylated SMAD3 (p-SMAD3), fibronectin (F/N), collagen IV (C-IV) and alpha-smooth muscle actin (α-SMA) expression was assessed via western blot and immunofluorescence. Lucigenin-enhanced chemiluminescence, and dihydroethidium (DHE) assessed NADPH oxidase activity and superoxide (SO), respectively. In iHMCs, a Pi transporter blocker (PFA) abrogated high Pi-induced AMPK inactivation, increase in NADPH oxidase-induced reactive oxygen species (ROS) levels, NOX4, p-SMAD3, α-SMA and C-IV expression. AMPK activation by AICAR, NOX4 silencing or NADPH oxidase blocker prevented high Pi-induced DHE levels, p-SMAD3, F/N, C-IV and α-SMA expression. AMPK inactivation with NOX4-induced ROS formation and transforming growth factor ß-1 (TGFß-1) signaling activation mediates high Pi-induced ECM accumulation in iHMCs. Maneuvers increasing AMPK or reducing NOX4 activity may contribute to renal protection under hyperphosphatemia.

Effects Of Partial Inhibition Of Respiratory Complex I On H2o 2 Production By Isolated Brain Mitochondria In Different Respiratory States.

The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.

Improvement Of The Physical Performance Is Associated With Activation Of No/pgc-1α/mttfa Signaling Pathway And Increased Protein Expressions Of Electron Transport Chain In Gastrocnemius Muscle From Rats Supplemented With L-arginine.

To examine the influence of l-arginine supplementation in combination with physical training on mitochondrial biomarkers from gastrocnemius muscle and its relationship with physical performance. Male Wistar rats were divided into four groups: control sedentary (SD), sedentary supplemented with l-arginine (SDLA), trained (TR) and trained supplemented with l-arginine (TRLA). Supplementation of l-arginine was administered by gavage (62.5mg/ml/day/rat). Physical training consisted of 60min/day, 5days/week, 0% grade, speed of 1.2km/h. The study lasted 8weeks. Skeletal muscle mitochondrial enriched fraction as well as cytoplasmic fractions were obtained for Western blotting and biochemical analyses. Protein expressions of transcriptor coactivator (PGC-1α), transcriptor factors (mtTFA), ATP synthase subunit c, cytochrome oxidase (COXIV), constitutive nitric oxide synthases (eNOS and nNOS), Cu/Zn-superoxide dismutase (SOD) and manganese-SOD (Mn-SOD) were evaluated. We also assessed in plasma: lipid profile, glycemia and malondialdehyde (MDA) levels. The nitrite/nitrate (NOx(-)) levels were measured in both plasma and cytosol fraction of the gastrocnemius muscle. 8-week l-arginine supplementation associated with physical training was effective in promoting greater tolerance to exercise that was accompanied by up-regulation of the protein expressions of mtTFA, PGC-1α, ATP synthase subunit c, COXIV, Cu/Zn-SOD and Mn-SOD. The upstream pathway was associated with improvement of NO bioavailability, but not in NO production since no changes in nNOS or eNOS protein expressions were observed. This combination would be an alternative approach for preventing cardiometabolic diseases given that in overt diseases a profound impairment in the physical performance of the patients is observed.

Theobromine Increases Nad⁺/sirt-1 Activity And Protects The Kidney Under Diabetic Conditions.

Reduction in sirtuin 1 (Sirt-1) is associated with extracellular matrix (ECM) accumulation in the diabetic kidney. Theobromine may reduce kidney ECM accumulation in diabetic rats. In the current study, we aimed to unravel, under diabetic conditions, the mechanism of kidney ECM accumulation induced by a reduction in Sirt-1 and the effect of theobromine in these events. In vitro, we used immortalized human mesangial cells (iHMCs) exposed to high glucose (HG; 30 mM), with or without small interfering RNA for NOX4 and Sirt-1. In vivo, spontaneously hypertensive rats (SHR) were rendered diabetic by means of streptozotocin and studied after 12 wk. The effects of treatment with theobromine were investigated under both conditions. HG leads to a decrease in Sirt-1 activity and NAD(+) levels in iHMCs. Sirt-1 activity could be reestablished by treatment with NAD(+), silencing NOX4, and poly (ADP-ribose) polymerase-1 (PARP-1) blockade, or with theobromine. HG also leads to a low AMP/ATP ratio, acetylation of SMAD3, and increased collagen IV, which is prevented by theobromine. Sirt-1 or AMPK blockade abolished these effects of theobromine. In diabetic SHR, theobromine prevented increases in albuminuria and kidney collagen IV, reduced AMPK, elevated NADPH oxidase activity and PARP-1, and reduced NAD(+) levels and Sirt-1 activity. These results suggest that in diabetes mellitus, Sirt-1 activity is reduced by PARP-1 activation and NAD(+) depletion due to low AMPK, which increases NOX4 expression, leading to ECM accumulation mediated by transforming growth factor (TGF)-β1 signaling. It is suggested that Sirt-1 activation by theobromine may have therapeutic potential for diabetic nephropathy.

Ensaio in vitro da enzima nitrato redutase e efeito da disponibilidade de nitrato e fosfato em variantes pigmentares de Hypnea musciformis (Wulfen) J. V. Lamour. (Gigartinales, Rhodophyta)

A enzima nitrato redutase (NR) catalisa a redução do nitrato a nitrito e controla a taxa de assimilação do nitrato. O ensaio in vitro da nitrato redutase foi otimizado para a linhagem selvagem (marrom, MA) e para a linhagem deficiente em ficoeritrina (verde-clara, VC) de Hypnea musciformis. As duas linhagens foram cultivadas em temperatura de 23 ± 2°C, fotoperíodo de 14 horas, irradiância de 60-90µmol fótons m-2s-1, e meio composto por água do mar esterilizada (30ups) enriquecida com a solução de von Stosch na concentração de 50% (VSES/2). As condições ótimas de ensaio para ambas as linhagens foram: 40µM de NADH; 10min de incubação do extrato bruto (EB) e 100µL de EB. A atividade ótima da NR ocorreu em 4 e 2mM de nitrato para a linhagem VC e MA, respectivamente. As linhagens VC e MA apresentaram, respectivamente, constante aparente de Michaelis-Menten (K M) para NADH de 0,2068 e 0,0837 µM, e K M para nitrato de 0,0492 e 0,0294mM. Os resultados indicam que a NR da linhagem MA tem maior afinidade pelo substrato do que a NR da linhagem VC de H. musciformis. Os experimentos para avaliar os efeitos da disponibilidade de nitrato (5 a 105µM) e nitrato e fosfato (0,5 a 25,5µM, com a relação N:P de 4:1) mostraram que a atividade da NR das linhagens VC e MA não aumentou com a adição de nitrato no meio, o que pode estar relacionado com o estado nutricional dessas algas. A atividade da NR foi maior nos tratamentos com adição de fosfato do que naqueles com adição de apenas nitrato, indicando que esse nutriente é importante para os processos metabólicos relacionados a atividade da NR.

O papel do polimorfismo funcional VNTR da região promotora do gene MAOA nos transtornos psiquiátricos

INTRODUÇÃO: Muitos estudos têm investigado a associação do polimorfismo VNTR (número variável de repetições em série) localizado na região promotora do gene da enzima monoamina oxidase A (MAOA) com alterações no comportamento humano e em diversos transtornos psiquiátricos. OBJETIVO: O objetivo do presente trabalho foi revisar a literatura sobre a participação desse polimorfismo funcional na modulação do comportamento humano para o desenvolvimento dos transtornos psiquiátricos. MÉTODO: A pesquisa foi realizada na literatura em inglês, de janeiro de 1998 a junho de 2009, disponível no Medline, Embase, Web of Science e na base de dados PsycInfo, utilizando os seguintes termos: "MAOA e comportamento humano" e "MAOA e psiquiatria". RESULTADOS: Foram encontrados 3.873 estudos. Desses, 109 foram selecionados e incluídos na revisão. Encontrou-se associação de alelos de baixa atividade do VNTR com transtorno de personalidade antissocial, transtorno de conduta, transtorno de déficit de atenção e hiperatividade, jogo patológico e dependência de substâncias. Alelos da alta atividade da MAOA foram associados a depressão, ansiedade, neuroticismo e anorexia nervosa. Não se encontrou associação entre polimorfismos da MAOA e esquizofrenia e transtorno bipolar. CONCLUSÃO: Os principais achados dão suporte ao papel do polimorfismo VNTR da região promotora do gene da MAOA em alguns transtornos psiquiátricos, apesar das divergências encontradas devidas às dificuldades metodológicas de estudos em genética. De modo geral, os estudos associam os alelos de baixa atividade da MAOA com comportamentos impulsivos e agressivos ("comportamentos hiperativos"), enquanto os alelos de alta atividade do gene são mais associados a "comportamentos hipoativos".

Molecular phylogeny of the Myzorhynchella Section of Anopheles (Nyssorhynchus) (Diptera: Culicidae): genetic support for recently described and resurrected species

Phylogenetic relationships among species of the Myzorhynchella Section of Anopheles (Nyssorhynchus) were investigated using the nuclear ribosomal DNA second internal transcribed spacer (ITS2), the nuclear whitegene and mitochondrial cytochrome oxidase subunit I (COI) regions. The recently described Anopheles pristinus and resurrected Anopheles guarani were also included in the study. Bayesian phylogenetic analyses found Anopheles parvus to be the most distantly related species within the Section, a finding that is consistent with morphology. An. pristinus and An. guarani were clearly resolved from Anopheles antunesi and Anopheles lutzii, respectively. An. lutzii collected in the same mountain range as the type locality were found within a strongly supported clade, whereas individuals from the southern state of Rio Grande do Sul, tentatively identified as An. lutzii based on adult female external morphology, were distinct from An. lutzii, An. antunesi and from each other, and may therefore represent two new sympatric species. A more detailed examination of An. lutzii sensu latoalong its known geographic range is recommended to resolve these anomalous relationships.

The development of an immobilized enzyme reactor containing glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi: the effect of species' specific differences on the immobilization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD+ from NADH. The production of NADH stimulated by D-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K-m) values determined for D-glyceraldehyde-3-phosphate and NAD(+) were K-m = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.

Blood Meal-Derived Heme Decreases ROS Levels in the Midgut of Aedes aegypti and Allows Proliferation of Intestinal Microbiota

The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS) generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF) mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox) reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme.

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

TRAINING ALTERS CARDIAC NEURON SIZES IN WISTAR RATS

The action of the parasympathetic nerves on the heart is made through a group of neurons located on the surface of the atria. This study evaluated the effect of a chronic training protocol on the number and sizes of the cardiac neurons of Wistar rats. Whole mount preparations of the atria of 12-month old male sedentary and trained rats (40 weeks of running on a treadmill 3 times a week, 16 m/min) were assessed for number and size (maximal cellular profile area) of the cardiac neurons. The cardiac neurons were ascertained by using the NADH-diaphorase technique that stains the cell bodies of the neurons in dark blue. The, number of cardiac neurons in the trained rats (P>0.05) did not change significantly. In the sedentary group there were small, medium sized and large neurons. However there was a notable increase in the percentage of small neurons in the rats submitted to the training compared to the sedentary group (P<0.05). Previous studies have shown that electrophysiologically, the small neurons are more easily excitable than the large neurons. It is possible that the results of the present work reflect an adaptation mechanism of the cardiac neurons presumably with the objective of increasing the excitability of the neurons for the vagal action and resulting facilitation of the sinusal bradycardia observed at rest and in the exercise. We concluded that the training affects significantly the size of the cardiac neurons in Wistar rats. (Biol.Sport 26.245-254, 2009)