Germinação de embriões zigóticos e desenvolvimento in vitro de coquinho-azedo

Este trabalho foi desenvolvido com objetivo de avaliar os efeitos da luz, das concentrações de sais MS e de sacarose sobre a germinação, in vitro, de embriões, e o desenvolvimento de plântulas de coquinho-azedo (Butia capitata (Mart.) Becc.). Para o preparo do meio de cultivo, foram utilizados sais MS suplementados com vitaminas, mio-inositol, caseína hidrolizada, carvão ativado, sacarose e ágar. Para avaliação do efeito da luminosidade, embriões zigóticos foram cultivados na presença e na ausência de luz. Diferentes concentrações de minerais MS (0; 25; 50; 75 e 100% em relação à formulação original do meio MS) foram testadas em associação com duas concentrações de sacarose ( de0 a 2%). Em ambos os experimentos, avaliaram-se, após 30 dias da inoculação, percentuais de oxidação, germinação e emissão de raízes e bainhas foliares. Os resultados obtidos mostraram que condição de luminosidade não afetou a germinação, porém, a ausência de luz favoreceu a emissão de raízes. Ocorreu o alongamento na ausência de sacarose, indicando a existência de reservas energéticas no embrião. A adição de sacarose proporcionou menores níveis de oxidação, favoreceu o alongamento e mostrou-se imprescindível para o desenvolvimento inicial das plântulas. Concentrações de sais entre 50 e 75% da concentração original do meio MS proporcionaram menores níveis de oxidação. A concentração de 75 % da concentração original de sais do meio MS proporcionou maior enraizamento.

A minimal version of a Protein Tyrosine Phosphatase

Phosphatase and tensin homologue (PTEN) protein belongs to the family of protein tyrosine phos-phatase. Mutations on the phosphatase and tensin homologue (PTEN) protein are highly observed in diverse types of human tumors, being mostly identified on the phosphatase domain of the protein. Although PTEN is a modular protein composed by a phosphatase domain and a C2 domain for mem-brane anchoring, this work aimed at developing a minimal version of PTEN´s phosphatase domain. The minimal version (Small Domain) comprises a 28 residue peptide, with the PTEN 8-mer catalytic peptide accommodated between a α-helix and β-turn as observed in PTEN native structure. Firstly, a de novo prediction of the Small Domain´s secondary structure was carried out by molecular modeling tools. The stability of the predicted structures were then evaluated by Molecular Dynamics. Automated molecular docking of PTEN natural substrate PIP3, its analogue (Inositol) and a PTEN inhibitor (L-tar-tare) were performed with the modeled structure, and PTEN used as a positive control. The gene en-coding for Small Domain was designed and cloned into an expression vector at N-terminal of Green Fluorescence Protein (GFP) encoding gene. The fusion protein was then expressed in Escherichia coli cells. Different expression conditions have been explored for the production of the fusion protein to minimize the formation of inclusion bodies.

Study of grapevine solute transporters involved in berry quality. A biochemical and molecular approach

Tese de Doutoramento em Biologia de Plantas

Metabolismo de lípidos y fosforilación proteica en Trypanosoma cruzi: su relación con la respuesta celular

Se propone un estudio en Trypanosoma cruzi , de aquellos procesos celulares que conducen a determinadas respuestas fisiológicas después de estímulos acoplados a receptores, tal como ocurre en eucariotas superiores. Debido a que dicha respuesta puede implicar cambios transitorios en la composición lipídica y en la fosforilación de proteínas se pretende continuar con el proyecto iniciado anteriormente, para dilucidar algunos aspectos de la regulación del ciclo del inositol fosfato. Para ello se caracterizará funcionalmente el ciclo en formas epimastigotes del parásito y se determinará el compromiso de los fosfogliceridos (específicamente los fosfonositidos) y lípidos neutros (diacilglicerol) con la respuesta celular frente a carbacol y al péptido sintético (1-40) que posee residuos del extremo amino terminal de la alfa D-globina de pollo. Con el propósito de determinar el papel que juega el IP3 como segundo mensajero en T. cruzi y sus consecuencias en la señal de calcio intracelular se estudiará el origen del incremento del ion después de los estímulos extracelulares mencionados anteriormente.

Lípido quinasas de raíz de tomate (Lycopersicum esculentum L.) bajo condiciones de estrés salino

Los tejidos en general, presentan al menos dos clases de sistemas transductores de la información a través de una membrana. Uno de ellos esta relacionado al AMPc, mientras que el otro induce a un rápido recambio de los fosfolípidos del inositol, así como una movilización del ión Ca2+. (...) Se tratará de optimizar las condiciones para estudiar la actividad de lípido quinasas en raíces de tomate ( Lycopersicum esculentum L.) cvs Pera y otras, para dilucidar si las mismas están de algún modo comprometidas en las respuestas fisiológicas de ese vegetal al estrés salino. (...) La existencia de vías de señales interconectadas en plantas, podría ayudar a explicar la manera por la que un pequeño grupo de fitohormonas determinarían una amplia variedad de respuestas celulares. Ahí estas podrían desempeñar un papel importante como sustancia señal en la cadena de transducción entre el estrés salino y las respuestas moleculares induciendo expresión génica y/o la acumulación de proteínas específicas u otros compuestos relacionados. Objetivo general: * Estudiar la actividad de lípido quinasas de raíz de tomate para dilucidar si son enzimas claves en el camino de transducción de señales que involucra fosfoinosítidos en plantas bajo condiciones de estrés salino. Objetivos específicos: * Estudiar la capacidad de las quinasas lipídas y diacilglicerol quinasa de raíz de tomate para transferir el fosfato del ATP a de los sustratos lipídicos endógenos. * Analizar la actividad de estas enzimas en raíces de tomate sometidas a estrés salino durante diferentes períodos de tiempo. * Iniciar la caracterización de la actividades de PI-k , y DG-k y estudias su posible relación con la respuesta celular a un estímulo externo, tanto en raíces controles como en raíces sometidas a estrés salino.

Metabolismo de fosfolípidos, señal de calcio y sus implicancias en la diferenciación de Trypanosoma cruzi. Phospholipids metabolism, calcium signal and its implications in the differentiation of Trypanosoma cruzi.

Trypanosoma cruzi es un protozoo primitivo agente causal de la enfermedad de Chagas. La transmisión de esta enfermedad depende tanto del desarrollo y de la diferenciación del microorganismo en el intestino del vector. Las diferentes formas del parásito se han adaptado a una serie de condiciones impuestas por los distintos ambientes en donde debió habitar. Esta capacidad de sobrevivir a medios externos tan variados está dada por la diversidad en las vías de transducción de señales en el parásito. T. cruzi se multiplica y diferencia (metaciclogénesis) en el recto de los triatominos. A este nivel, los parásitos se enfrentan a un incremento en la osmolaridad causado por un elevado contenido de NaCl en la orina. En nuestro laboratorio se observó que diferentes estímulos son capaces de producir incrementos en los niveles de IP3 y de Ca2+ intracelular, consecuencia de la activación del ciclo del inositol fosfato, y activación de fosfolipasa D (PLD) y fosfatidilinositol 3 quinasa (PI3K). En un medio carente de Na+ los epimastigotes estimulados con carbacol, mostraron una señal de calcio disminuida mientras que la acumulación de IP3 no se modificó. Además, esta señal se incrementó en presencia de PMA, activador de proteína quinasa C, mientras que la acumulación de IP3 se anuló completamente. Estos resultados indujeron a pensar en un mecanismo alternativo y/o paralelo a IP3 en la liberación de Ca2+, en el cual la presencia de un intercambiador Na+/H+ favorecería la liberación del ion desde organelas acídicas. Es conocido que la señal de calcio es requerida para la metaciclogénesis, y que esta señal es independiente del Ca2+ extracelular (Lammel y col. 1996, Marchesini y col., 2002). De este modo se propone que "los epimastigotes de T. cruzi utilizan como elementos conservados a lo largo de la evolución a los elementos del ciclo del inositol fosfato, uno de los sistemas de transducción de señales más antiguo, para responder a estímulos que inducen la diferenciación del parásito". Por lo tanto, para el desarrollo de este proyecto se propone determinar la presencia de un RcIP3 en epimastigotes y conocer su compromiso en la liberación de Ca2+ desde reservorios intracelulares. Además, establecer si un intercambiador Na+/H+ en membrana de acidocalcisomas estaría relacionado con la señal de calcio intracelular y su posible regulación por proteina quinasa C y A (PKC y PKA, respectivamente). Por otro lado, para dilucidar la implicancia de estos mecanismos en el proceso de metaciclogénesis, se propone estudiar la activación del intercambiador Na+/H+ y la señal de calcio en condiciones de hiperosmolaridad, tal como ocurre en el recto del triatomino. Ademas, ya que el proceso de diferenciación involucra una reorganización de los microtubulos del citoesqueleto se pretende estudiar el compromiso del metabolismo de fosfolípidos y tubulina en procesos que contribuyen a la inducción de la metaciclogenesis. El alcance de los objetivos mencionados ayudará a dilucidar la presencia de componentes tales como RcIP3 y el intercambiador Na+/H+ involucrados en la señalización del ion bivalente. Por otro lado, se espera demostrar que los isotipos de tubulina encontrados en T. cruzi cambien en cantidad relativa y nivel de expresión cuando los epimastigotes sean estimulados con posibles inductores de la diferenciación. Además, se espera observar simultáneamente un aumento en la actividad de dos enzimas relacionadas con la reorganización de microtúbulos: PI-3K y PLD. En tal caso, y para comprobar su implicancia en el proceso, se espera que la inhibición de tales enzimas sea capaz de revertir el efecto producido por los estímulos. Como la PLC se expresa principalmente en las forma epimastigotes mas que en los tripomastigotes (forma infectiva), la señal de Ca2+ inducida por IP3 se relacionaría con la capacidad del parásito para responder a ciertos cambios de pH y osmolaridad que enfrenta el microorganismo en el tracto digestivo del insecto vector.

Cultura in vitro de sementes e anteras de Sesamum indicum L.

Sementes de gergelin (Sesamum indicum L.) foram cultivadas in vitro em meio de cultura de Wetherall contendo 0,5 mg/l de 2,4-D e em seguida transferidas para meio de Murashige e Skoog (MS) contendo 0,1 mg/l de 2,4-D e 100 mg/l de inositol. Ambos, 2,4-D e inositol mostraram-se ser necessários para o desenvolvimento de calos a partir de sementes, do mesmo modo que para o contínuo crescimento dos meios em cultura. Foram também obtidos calos de explantes de anteras, cotiledones e de hipocotilo de Sesamum utilizando-se o meio MS com a ocorrência de estruturas globulares.

Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation.

We have suggested previously that both the negatively and positively charged residues of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif play an important role in the activation process of the alpha(1b)-adreneric receptor (AR). In this study, R143 of the E/DRY sequence in the alpha(1b)-AR was mutated into several amino acids (Lys, His, Glu, Asp, Ala, Asn, and Ile). The charge-conserving mutation of R143 into lysine not only preserved the maximal agonist-induced response of the alpha(1b)-AR, but it also conferred high degree of constitutive activity to the receptor. Both basal and agonist-induced phosphorylation levels were significantly increased for the R143K mutant compared with those of the wild-type receptor. Other substitutions of R143 resulted in receptor mutants with either a small increase in constitutive activity (R143H and R143D), impairment (R143H, R143D), or complete loss of receptor-mediated response (R143E, R143A, R143N, R143I). The R413E mutant displayed a small, but significant increase in basal phosphorylation despite being severely impaired in receptor-mediated response. Interestingly, all the arginine mutants displayed increased affinity for agonist binding compared with the wild-type alpha(1b)-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants using the allosteric ternary complex model in conjunction with the results of molecular dynamics simulations on the receptor models support the hypothesis that mutations of R143 can drive the isomerization of the alpha(1b)-AR into different states, highlighting the crucial role of this residue in the activation process of the receptor.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Caffeine consumption attenuates neurochemical modifications in the hippocampus of streptozotocin-induced diabetic rats.

Type 1 diabetes can affect hippocampal function triggering cognitive impairment through unknown mechanisms. Caffeine consumption prevents hippocampal degeneration and memory dysfunction upon different insults and is also known to affect peripheral glucose metabolism. Thus we now characterized glucose transport and the neurochemical profile in the hippocampus of streptozotocin-induced diabetic rats using in vivo(1)H NMR spectroscopy and tested the effect of caffeine consumption thereupon. We found that hippocampal glucose content and transport were unaltered in diabetic rats, irrespective of caffeine consumption. However diabetic rats displayed alterations in their hippocampal neurochemical profile, which were normalized upon restoration of normoglycaemia, with the exception of myo-inositol that remained increased (36 +/- 5%, p < 0.01 compared to controls) likely reflecting osmolarity deregulation. Compared to controls, caffeine-consuming diabetic rats displayed increased hippocampal levels of myo-inositol (15 +/- 5%, p < 0.05) and taurine (23 +/- 4%, p < 0.01), supporting the ability of caffeine to control osmoregulation. Compared to controls, the hippocampus of diabetic rats displayed a reduced density of synaptic proteins syntaxin, synaptophysin and synaptosome-associated protein of 25 kDa (in average 18 +/- 1%, p < 0.05) as well increased glial fibrillary acidic protein (20 +/- 5%, p < 0.05), suggesting synaptic degeneration and astrogliosis, which were prevented by caffeine consumption. In conclusion, neurochemical alterations in the hippocampus of diabetic rats are not related to defects of glucose transport but likely reflect osmoregulatory adaptations caused by hyperglycemia. Furthermore, caffeine consumption affected this neurochemical adaptation to high glucose levels, which may contribute to its potential neuroprotective effects, namely preventing synaptic degeneration and astrogliosis.

PPARβ/δ prevents endoplasmic reticulum stress-associated inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism.

AIM/HYPOTHESIS: Endoplasmic reticulum (ER) stress, which is involved in the link between inflammation and insulin resistance, contributes to the development of type 2 diabetes mellitus. In this study, we assessed whether peroxisome proliferator-activated receptor (PPAR)β/δ prevented ER stress-associated inflammation and insulin resistance in skeletal muscle cells. METHODS: Studies were conducted in mouse C2C12 myotubes, in the human myogenic cell line LHCN-M2 and in skeletal muscle from wild-type and PPARβ/δ-deficient mice and mice exposed to a high-fat diet. RESULTS: The PPARβ/δ agonist GW501516 prevented lipid-induced ER stress in mouse and human myotubes and in skeletal muscle of mice fed a high-fat diet. PPARβ/δ activation also prevented thapsigargin- and tunicamycin-induced ER stress in human and murine skeletal muscle cells. In agreement with this, PPARβ/δ activation prevented ER stress-associated inflammation and insulin resistance, and glucose-intolerant PPARβ/δ-deficient mice showed increased phosphorylated levels of inositol-requiring 1 transmembrane kinase/endonuclease-1α in skeletal muscle. Our findings demonstrate that PPARβ/δ activation prevents ER stress through the activation of AMP-activated protein kinase (AMPK), and the subsequent inhibition of extracellular-signal-regulated kinase (ERK)1/2 due to the inhibitory crosstalk between AMPK and ERK1/2, since overexpression of a dominant negative AMPK construct (K45R) reversed the effects attained by PPARβ/δ activation. CONCLUSIONS/INTERPRETATION: Overall, these findings indicate that PPARβ/δ prevents ER stress, inflammation and insulin resistance in skeletal muscle cells by activating AMPK.

The Role of Protein Kinases in Antigen-activation of Peripheral Blood Mononuclear Cells of Schistosoma mansoni Infected Individuals

T cell recognition of antigens displayed on the surface of antigen presenting cell results in rapid activation of protein tyrosine kinases and kinase C. This process leads to second messengers, such as inositol phosphates and diacylgycerol, and phosphorylation of multiple proteins. The role of different protein kinases in the activation of peripheral blood mononuclear cells (PBMC) from Schistosoma mansoni infected individuals was evaluated using genistein and H-7, specific inhibitors of protein tyrosine kinase and kinase C, respectively. Our results showed that proliferation in response to soluble egg antigen or adult worm antigen preparation of S. mansoni was reduced when PBMC were cultured in presence of protein kinase inhibitors. Using these inhibitors on in vitro granuloma reaction, we also observed a marked reduction of granuloma index. Taken together, our results suggest that S. mansoni antigen activation of PBMC involves protein kinases activity

Truncation of the receptor carboxyl terminus impairs agonist-dependent phosphorylation and desensitization of the alpha 1B-adrenergic receptor.

The alpha 1B-adrenergic receptor (alpha 1BAR) and its truncated mutant T368 lacking the last 147 amino acids were stably expressed in Rat1 fibroblasts. The wild type alpha 1BAR was rapidly phosphorylated upon exposure to the agonist epinephrine as well as to phorbol ester as assessed by immunoprecipitation of the receptor with antiserum raised against its amino-terminal portion. Exposure of cells expressing the wild type alpha 1BAR to epinephrine resulted also in rapid homologous desensitization of receptor-mediated response on polyphosphoinositide hydrolysis. On the other hand, truncation of the serine- and threonine-rich carboxyl portion of the alpha 1BAR abolished agonist-induced phosphorylation and greatly impaired homologous desensitization of the receptor. The truncated receptor T368 could undergo agonist-induced decrease of cell surface receptors but to a lesser extent, as compared with the wild type alpha 1BAR. These results demonstrate that the carboxyl portion of the alpha 1BAR plays a crucial role in the regulation of receptor function. They also suggest a strong relationship between agonist-induced phosphorylation and desensitization of the alpha 1BAR, which were both insensitive to the inhibitor of protein kinase C RO-318220. Our findings support the emerging hypothesis that the biochemical mechanisms involved in rapid agonist-dependent regulation of G protein-coupled receptors, which activate polyphosphoinositide hydrolysis, do not primarily involve protein kinase C.

The C57BL/6J Mouse Exhibits Sporadic Congenital Portosystemic Shunts.

C57BL/6 mice are the most widely used strain of laboratory mice. Using in vivo proton Magnetic Resonance Spectroscopy ((1)H MRS), we have repeatedly observed an abnormal neurochemical profile in the brains of both wild-type and genetically modified mice derived from the C57BL/6J strain, consisting of a several fold increase in cerebral glutamine and two fold decrease in myo-inositol. This strikingly abnormal neurochemical "phenotype" resembles that observed in chronic liver disease or portosystemic shunting and appeared to be independent of transgene, origin or chow and was not associated with liver failure. As many as 25% of animals displayed the abnormal neurochemical profile, questioning the reliability of this model for neurobiology. We conducted an independent study to determine if this neurochemical profile was associated with portosystemic shunting. Our results showed that 100% of the mice with high brain glutamine displayed portosystemic shunting by concomitant portal angiography while all mice with normal brain glutamine did not. Since portosystemic shunting is known to cause alterations in gene expression in many organs including the brain, we conclude that portosystemic shunting may be the most significant problem associated with C57BL/6J inbreeding both for its effect on the central nervous system and for its systemic repercussions.

Characterization of metabolites in infiltrating gliomas using ex vivo &supl;H high-resolution magic angle spinning spectroscopy.

Gliomas are routinely graded according to histopathological criteria established by the World Health Organization. Although this classification can be used to understand some of the variance in the clinical outcome of patients, there is still substantial heterogeneity within and between lesions of the same grade. This study evaluated image-guided tissue samples acquired from a large cohort of patients presenting with either new or recurrent gliomas of grades II-IV using ex vivo proton high-resolution magic angle spinning spectroscopy. The quantification of metabolite levels revealed several discrete profiles associated with primary glioma subtypes, as well as secondary subtypes that had undergone transformation to a higher grade at the time of recurrence. Statistical modeling further demonstrated that these metabolomic profiles could be differentially classified with respect to pathological grading and inter-grade conversions. Importantly, the myo-inositol to total choline index allowed for a separation of recurrent low-grade gliomas on different pathological trajectories, the heightened ratio of phosphocholine to glycerophosphocholine uniformly characterized several forms of glioblastoma multiforme, and the onco-metabolite D-2-hydroxyglutarate was shown to help distinguish secondary from primary grade IV glioma, as well as grade II and III from grade IV glioma. These data provide evidence that metabolite levels are of interest in the assessment of both intra-grade and intra-lesional malignancy. Such information could be used to enhance the diagnostic specificity of in vivo spectroscopy and to aid in the selection of the most appropriate therapy for individual patients.