Ammonia-induced toxicity in developing brain cells and strategies for neuroprotection

RESUME L'hyperammonémie est particulièrement toxique pour le cerveau des jeunes patients et entraîne une atrophie corticale, un élargissement des ventricules et des défauts de myélinisation, responsables de retards mentaux et développementaux. Les traitements actuels se limitent à diminuer le plus rapidement possible le taux d'ammoniaque dans l'organisme. L'utilisation de traitements neuroprotecteurs pendant les crises d'hyperammonémie permettrait de contrecarrer les effets neurologiques de l'ammoniaque et de prévenir l'apparition des troubles neurologiques. Au cours de cette thèse, nous avons testé trois stratégies de neuroprotection sur des cultures de cellules en agrégats issues du cortex d'embryons de rats et traitées à l'ammoniaque. - Nous avons tout d'abord testé si l'inhibition de protéines intracellulaires impliquées dans le déclenchement de la mort cellulaire pouvait protéger les cellules de la toxicité de l'ammoniaque. Nous avons montré que L'exposition à l'ammoniaque altérait la viabilité des neurones et des oligodendrocytes, et activait les caspases, la calpaïne et la kinase-5 dépendante des cyclines (cdk5) associée à son activateur p25. Alors que l'inhibition pharmacologique des caspases et de la calpaïne n'a pas permis de protéger les cellules cérébrales, un inhibiteur de la cdk5, appelé roscovitine, a réduit significativement la mort neuronale. L'inhibition de la cdk5 semble donc être une stratégie thérapeutique prometteuse pour prévenir 1es effets toxiques de 1'ammoniaque sur les neurones. - Nous avons ensuite étudié les mécanismes neuroprotecteurs déclenchés par le cerveau en réponse à la toxicité de l'ammoniaque. Nous avons montré que l'ammoniaque induisait la synthèse du facteur neurotrophique ciliaire (CNTF) par les astrocytes, via l'activation de la protéine kinase (MIAPK) p38. D'autre part, l'ajout de CNTF a permis de protéger les oligodendrocytes mais pas les neurones des cultures exposées à l'ammoniaque, via les voies de signalisations JAK/STAT, SAPK/JNK et c-jun. - Dans une dernière partie, nous avons voulu contrecarrer, par l'ajout de créatine, le déficit énergétique cérébral induit par l'ammoniaque. La créatine a permis de protéger des cellules de type astrocytaire mais pas les cellules cérébrales en agrégats. Cette thèse amis en évidence que les stratégies de neuroprotection chez les patients hyperammonémiques nécessiteront de cibler plusieurs voies de signalisation afin de protéger tous les types cellulaires du cerveau. Summary : In pediatric patients, hyperammonemia is mainly caused by urea cycle disorders or other inborn errors of metabolism, and leads to neurological injury with cortical atrophy, ventricular enlargement and demyelination. Children rescued from neonatal hyperammonemia show significant risk of mental retardation and developmental disabilities. The mainstay of therapy is limited to ammonia lowering through dietary restriction and alternative pathway treatments. However, the possibility of using treatments in a neuroprotective goal may be useful to improve the neurological outcome of patients. Thus, the main objective of this work was to investigate intracellular and extracellular signaling pathways altered by ammonia tonicity, so as to identify new potential therapeutic targets. Experiments were conducted in reaggregated developing brain cell cultures exposed to ammonia, as a model for the developing CNS of hyperammonemic young patients. Theses strategies of neuroprotection were tested: - The first strategy consisted in inhibiting intracellular proteins triggering cell death. Our data indicated that ammonia exposure altered the viability of neurons and oligodendrocytes. Apoptosis and proteins involved in the trigger of apoptosis, such as caspases, calpain and cyclin-dependent kinase-5 (cdk5) with its activator p25, were activated by ammonia exposure. While caspases and calpain inhibitors exhibited no protective effects, roscovitine, a cdk5 inhibitor, reduced ammonia-induced neuronal death. This work revealed that inhibition of cdk5 seems a promising strategy to prevent the toxic effects of ammonia on neurons. - The second strategy consisted in mimicking, the endogenous protective mechanisms triggered by ammonia in the brain. Ammonia exposure caused an increase of the ciliary neurotrophic factor (CNTF) expression, through the activation of the p38 mitogen-activated protein kinase (MAPK) in astrocytes. Treatment of cultures exposed to ammonia with exogenous CNTF demonstrated strong protective effects on oligodendrocytes but not on neurons. These protective effects seemed to involve JAK/STAT, SAPK/JNK and c-jun proteins. - The third strategy consisted in preventing the ammonia-induced cerebral energy deficit with creatine. Creatine treatment protected the survival of astrocyte-like cells through MAPKs pathways. In contrast, it had no protective effects in reaggregated developing brain cell cultures exposed to ammonia. The present study suggests that neuroprotective strategies should optimally be directed at multiple targets to prevent ammonia-induced alterations of the different brain cell types.

Timing, location and crop species influence the magnitude of amelioration of aluminum toxicity by magnesium

The protective effect of cations, especially Ca and Mg, against aluminum (Al) rhizotoxicity has been extensively investigated in the last decades. The mechanisms by which the process occurs are however only beginning to be elucidated. Six experiments were carried out here to characterize the protective effect of Mg application in relation to timing, location and crop specificity: Experiment 1 - Protective effect of Mg compared to Ca; Experiment 2 - Protective effect of Mg on distinct root classes of 15 soybean genotypes; Experiment 3 - Effect of timing of Mg supply on the response of soybean cvs. to Al; Experiment 4 - Investigating whether the Mg protective effect is apoplastic or simplastic using a split-root system; Experiment 5 - Protective effect of Mg supplied in solution or foliar spraying, and Experiment 6 - Protective effect of Mg on Al rhizotoxicity in other crops. It was found that the addition of 50 mmol L-1 Mg to solutions containing toxic Al increased Al tolerance in 15 soybean cultivars. This caused soybean cultivars known as Al-sensitive to behave as if they were tolerant. The protective action of Mg seems to require constant Mg supply in the external medium. Supplying Mg up to 6 h after root exposition to Al was sufficient to maintain normal soybean root growth, but root growth was not recovered by Mg addition 12 h after Al treatments. Mg application to half of the root system not exposed to Al was not sufficient to prevent Al toxicity on the other half exposed to Al without Mg in rooting medium, indicating the existence of an external protection mechanism of Mg. Foliar spraying with Mg also failed to decrease Al toxicity, indicating a possible apoplastic role of Mg. The protective effect of Mg appeared to be soybean-specific since Mg supply did not substantially improve root elongation in sorghum, wheat, corn, cotton, rice, or snap bean when grown in the presence of toxic Al concentrations.

Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity.

Eukaryotes contain inorganic polyphosphate (polyP) and acidocalcisomes, which sequester polyP and store amino acids and divalent cations. Why polyP is sequestered in dedicated organelles is not known. We show that polyP produced in the cytosol of yeast becomes toxic. Reconstitution of polyP translocation with purified vacuoles, the acidocalcisomes of yeast, shows that cytosolic polyP cannot be imported, whereas polyP produced by the vacuolar transporter chaperone (VTC) complex, an endogenous vacuolar polyP polymerase, is efficiently imported and does not interfere with growth. PolyP synthesis and import require an electrochemical gradient, probably as a driving force for polyP translocation. VTC exposes its catalytic domain to the cytosol and carries nine vacuolar transmembrane domains. Mutations in the VTC transmembrane regions, which are likely to constitute the translocation channel, block not only polyP translocation but also synthesis. Given that they are far from the cytosolic catalytic domain of VTC, this suggests that the VTC complex obligatorily couples synthesis of polyP to its import in order to avoid toxic intermediates in the cytosol. Sequestration of otherwise toxic polyP might be one reason for the existence of acidocalcisomes in eukaryotes.

[(18)F]FDG-PET Standard Uptake Value as a Metabolic Predictor of Bone Marrow Response to Radiation: Impact on Acute and Late Hematological Toxicity in Cervical Cancer Patients Treated With Chemoradiation Therapy.

PURPOSE: To quantify the relationship between bone marrow (BM) response to radiation and radiation dose by using (18)F-labeled fluorodeoxyglucose positron emission tomography [(18)F]FDG-PET standard uptake values (SUV) and to correlate these findings with hematological toxicity (HT) in cervical cancer (CC) patients treated with chemoradiation therapy (CRT). METHODS AND MATERIALS: Seventeen women with a diagnosis of CC were treated with standard doses of CRT. All patients underwent pre- and post-therapy [(18)F]FDG-PET/computed tomography (CT). Hemograms were obtained before and during treatment and 3 months after treatment and at last follow-up. Pelvic bone was autosegmented as total bone marrow (BMTOT). Active bone marrow (BMACT) was contoured based on SUV greater than the mean SUV of BMTOT. The volumes (V) of each region receiving 10, 20, 30, and 40 Gy (V10, V20, V30, and V40, respectively) were calculated. Metabolic volume histograms and voxel SUV map response graphs were created. Relative changes in SUV before and after therapy were calculated by separating SUV voxels into radiation therapy dose ranges of 5 Gy. The relationships among SUV decrease, radiation dose, and HT were investigated using multiple regression models. RESULTS: Mean relative pre-post-therapy SUV reductions in BMTOT and BMACT were 27% and 38%, respectively. BMACT volume was significantly reduced after treatment (from 651.5 to 231.6 cm(3), respectively; P<.0001). BMACT V30 was significantly correlated with a reduction in BMACT SUV (R(2), 0.14; P<.001). The reduction in BMACT SUV significantly correlated with reduction in white blood cells (WBCs) at 3 months post-treatment (R(2), 0.27; P=.04) and at last follow-up (R(2), 0.25; P=.04). Different dosimetric parameters of BMTOT and BMACT correlated with long-term hematological outcome. CONCLUSIONS: The volumes of BMTOT and BMACT that are exposed to even relatively low doses of radiation are associated with a decrease in WBC counts following CRT. The loss in proliferative BM SUV uptake translates into low WBC nadirs after treatment. These results suggest the potential of intensity modulated radiation therapy to spare BMTOT to reduce long-term hematological toxicity.

Mobilidade de P, Cu e Zn em colunas de solo sob sistema de semeadura direta submetido às adubações mineral e orgânica

A retenção, no perfil do solo, de elementos minerais aplicados em sua superfície é fundamental para a manutenção da qualidade da água. Nesse sentido, o objetivo deste trabalho foi avaliar o efeito da aplicação de dejeto líquido de suíno e adubo mineral na lixiviação do P, Cu e Zn em colunas indeformadas de solo (25 cm de altura e 20,3 cm de diâmetro) retiradas de uma área de Latossolo Vermelho eutroférrico típico sob semeadura direta. A água foi aplicada nas colunas de forma ininterrupta, na quantidade correspondente a três volumes da porosidade total do solo (10.020 cm³), sendo a coleta e análise dos elementos subdivididas em cinco frações. Cada volume de poros (3.340 cm³) corresponde a um turno de água percolada, e o primeiro volume foi fracionado em três coletas. A concentração dos elementos no solo foi determinada nas camadas de 0 a 2,5, 2,5 a 10 e 10 a 20 cm. A retenção de P, Cu, e Zn no solo, em relação ao aplicado via dejeto líquido de suíno, foi de 99,6, 98,8 e 100 %, respectivamente. Para o P aplicado via adubo mineral, a retenção foi de 89,7 %. Independentemente do tipo de adubo, as concentrações de P, Cu e Zn no solo foram maiores na camada de 0 a 2,5 cm. Esses resultados indicam o baixo potencial de lixiviação desses elementos, o que sugere ser importante o uso de práticas de manejo e conservação do solo e da água que proporcionem a contenção e a infiltração do escoamento superficial.

Hysteresis in a system driven by either generalized force or displacement variables: martensitic phase transition in single-crystalline Cu-Zn-Al

We report on experiments aimed at comparing the hysteretic response of a Cu-Zn-Al single crystal undergoing a martensitic transition under strain-driven and stress-driven conditions. Strain-driven experiments were performed using a conventional tensile machine while a special device was designed to perform stress-driven experiments. Significant differences in the hysteresis loops were found. The strain-driven curves show reentrant behavior yield point which is not observed in the stress-driven case. The dissipated energy in the stress-driven curves is larger than in the strain-driven ones. Results from recently proposed models qualitatively agree with experiments.

Teores de Fe, Mn, Zn, Cu, Ni E Co em solos de referência de Pernambuco

Metais pesados formam um grupo de elementos com particularidades relevantes e de ocorrência natural no ambiente, como elementos acessórios na constituição de rochas. Esses elementos, apesar de associados à toxidez, exigem tratamento diferenciado em relação aos xenobióticos, uma vez que diversos metais possuem essencialidade (Fe, Mn, Cu, Zn e Ni) e benefício (Co) comprovados para as plantas. Nesse contexto, o objetivo deste trabalho foi determinar os teores naturais dos metais Fe, Mn, Zn, Ni, Cu e Co nos solos de referência de Pernambuco. Foram coletadas amostras de solo nas três regiões fisiográficas (Zona da Mata, Agreste e Sertão), dos dois primeiros horizontes dos 35 solos de referência do Estado de Pernambuco. A digestão das amostras baseou-se no método 3051A (USEPA, 1998), e a determinação foi efetuada em ICP-OES. Correlações significativas foram estabelecidas entre os metais e entre estes e a fração argila do solo, em ambos os horizontes, indicando a associação comum da maioria dos metais com solos mais argilosos. A maioria dos solos apresentou teores de Fe, Mn, Zn, Cu, Ni e Co menores que os de solos de outras regiões do País, com litologia mais máfica, o que corrobora o fato de que os teores desses elementos são mais diretamente relacionados aos minerais Fe-magnesianos. Os resultados indicam baixo potencial dos solos de Pernambuco em liberar Cu, Co e Ni para plantas, enquanto deficiências de Zn, Fe e Mn são menos prováveis. Os teores naturais de Fe, Mn, Zn, Cu, Ni e Co determinados podem ser utilizados como base para definição dos Valores de Referência de Qualidade para os solos de Pernambuco, de acordo com o preconizado pela legislação nacional.

State equation for shape-memory alloys. Aplication to Cu-Zn-Al

We deal with the hysteretic behavior of partial cycles in the two¿phase region associated with the martensitic transformation of shape¿memory alloys. We consider the problem from a thermodynamic point of view and adopt a local equilibrium formalism, based on the idea of thermoelastic balance, from which a formal writing follows a state equation for the material in terms of its temperature T, external applied stress ¿, and transformed volume fraction x. To describe the striking memory properties exhibited by partial transformation cycles, state variables (x,¿,T) corresponding to the current state of the system have to be supplemented with variables (x,¿,T) corresponding to points where the transformation control parameter (¿¿ and/or T) had reached a maximum or a minimum in the previous thermodynamic history of the system. We restrict our study to simple partial cycles resulting from a single maximum or minimum of the control parameter. Several common features displayed by such partial cycles and repeatedly observed in experiments lead to a set of analytic restrictions, listed explicitly in the paper, to be verified by the dissipative term of the state equation, responsible for hysteresis. Finally, using calorimetric data of thermally induced partial cycles through the martensitic transformation in a Cu¿Zn¿Al alloy, we have fitted a given functional form of the dissipative term consistent with the analytic restrictions mentioned above.

Preisach modeling of hysteresis for a pseudoelastic Cu-Zn-Al single crystal

Stress-strain trajectories associated with pseudoelastic behavior of a Cu¿19.4 Zn¿13.1 Al (at.%) single crystal at room temperature have been determined experimentally. For a constant cross-head speed the trajectories and the associated hysteresis behavior are perfectly reproducible; the trajectories exhibit memory properties, dependent only on the values of return points, where transformation direction is reverted. An adapted version of the Preisach model for hysteresis has been implemented to predict the observed trajectories, using a set of experimental first¿order reversal curves as input data. Explicit formulas have been derived giving all trajectories in terms of this data set, with no adjustable parameters. Comparison between experimental and calculated trajectories shows a much better agreement for descending than for ascending paths, an indication of a dissymmetry between the dissipation mechanisms operative in forward and reverse directions of martensitic transformation.

Elastic constants of the beta-Cu-Zn alloy system: A Monte Carlo study

Monte Carlo calculations of the isothermal elastic constants of the beta-CuxZn1-x alloy system as a function of the composition have been carried out. We assume the atoms interact via a two-body Morse potential function and use numerical values for the potential parameters evaluated taking into account experimental data. We find a quite good agreement between our results and the expected experimental behavior.

Comparação de métodos convencional e alternativo para determinar Cu, Fe, Mn e Zn em extratos de solos obtidos com solução de mehlich-1

Nos laboratórios brasileiros, a determinação de micronutrientes para estudos de fertilidade de solos é realizada por extração com solução de Mehlich-1 (M-1), utilizando-se uma razão solo: extrator de 1:5 m/v e posterior quantificação por espectrometria de absorção atômica (AAS). No entanto, muitos laboratórios também empregam razão solo:extrator M-1 de 1:10 m/v para determinar macronutrientes e, ou, quantificar por espectrometria de emissão óptica com plasma indutivamente acoplado (ICP OES), em substituição ao AAS. Como estudos comparativos entre as concentrações de micronutrientes obtidos por esses métodos alternativos são escassos, o objetivo deste trabalho foi investigar se essas diferentes condições experimentais gerariam diferentes resultados. Os resultados foram estatisticamente tratados e revelaram que as concentrações de Fe e Mn, utilizando-se ambas as razões de solo:extrator M-1 (1:5 e 1:10 m/v) e de Zn, na razão solo:extrator M-1 de 1:10 m/v, foram estatisticamente iguais, enquanto as concentrações de Cu em ambas as razões solo:extrator M-1 e de Zn na razão solo:extrator de 1:5 m/v foram diferentes, quando as duas técnicas de quantificação (ICP OES e AAS) foram comparadas. Também, todas as concentrações de Cu, Fe, Mn e Zn obtidas utilizando razão solo:extrator M-1 de 1:5 m/v foram diferentes daquelas encontradas com a razão de 1:10 m/v, independentemente da técnica de quantificação. A maioria dessas concentrações foi maior quando se usou a razão de 1:5 m/v e alterou a classe de interpretação dos teores. Portanto, não recomenda-se o uso da razão solo:extrator de 1:5 para as extrações dos micronutrientes Cu, Fe, Mn e Zn com solução M-1 de amostras de solo.

Arsenic toxicity in Acacia mangium willd. and mimosa Caesalpiniaefolia benth. seedlings

Acacia mangium and Mimosa caesalpiniaefolia are fast-growing woody fabaceous species that might be suitable for phytoremediation of arsenic (As)-contaminated sites. To date, few studies on their tolerance to As toxicity have been published. Therefore, this study assessed As toxicity symptoms in A. mangium and M. caesalpiniaefolia seedlings under As stress in a greenhouse. Seedlings of Acacia mangium and M. caesalpiniaefolia were grown for 120 d in an Oxisol-sand mixture with 0, 50, 100, 200, and 400 mg kg-1 As, in four replications in four randomized blocks. The plants were assessed for visible toxicity symptoms, dry matter production, shoot/root ratio, root anatomy and As uptake. Analyses of variance and regression showed that the growth of A. mangium and M. caesalpiniaefolia was severely hindered by As, with a reduction in dry matter production of more than 80 % at the highest As rate. The root/shoot ratio increased with increasing As rates. At a rate of 400 mg kg-1 As, whitish chlorosis appeared on Mimosa caesalpiniaefolia seedlings. The root anatomy of both species was altered, resulting in cell collapse, death of root buds and accumulation of phenolic compounds. Arsenic concentration was several times greater in roots than in shoots, with more than 150 and 350 mg kg-1 in M. caesalpiniaefolia and A. mangium roots, respectively. These species could be suitable for phytostabilization of As-contaminated sites, but growth-stimulating measures should be used.

Electrical and optical properties of Zn-In-Sn-O transparent conducting thin films

Indium tin oxide (ITO) is one of the widely used transparent conductive oxides (TCO) for application as transparent electrode in thin film silicon solar cells or thin film transistors owing to its low resistivity and high transparency. Nevertheless, indium is a scarce and expensive element and ITO films require high deposition temperature to achieve good electrical and optical properties. On the other hand, although not competing as ITO, doped Zinc Oxide (ZnO) is a promising and cheaper alternative. Therefore, our strategy has been to deposit ITO and ZnO multicomponent thin films at room temperature by radiofrequency (RF) magnetron co-sputtering in order to achieve TCOs with reduced indium content. Thin films of the quaternary system Zn-In-Sn-O (ZITO) with improved electrical and optical properties have been achieved. The samples were deposited by applying different RF powers to ZnO target while keeping a constant RF power to ITO target. This led to ZITO films with zinc content ratio varying between 0 and 67%. The optical, electrical and morphological properties have been thoroughly studied. The film composition was analysed by X-ray Photoelectron Spectroscopy. The films with 17% zinc content ratio showed the lowest resistivity (6.6 × 10 - 4 Ω cm) and the highest transmittance (above 80% in the visible range). Though X-ray Diffraction studies showed amorphous nature for the films, using High Resolution Transmission Electron Microscopy we found that the microstructure of the films consisted of nanometric crystals embedded in a compact amorphous matrix. The effect of post deposition annealing on the films in both reducing and oxidizing atmospheres were studied. The changes were found to strongly depend on the zinc content ratio in the films.