Estudo estrutural por RMN do peptídeo policatiônico polybine I de veneno da vespa social Polybia paulista

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Hyperalgesic and edematogenic effects of Secapin-2, a peptide isolated from Africanized honeybee (Apis mellifera) venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Leuconostoc mesenteroides SJRP55: a bacteriocinogenic strain isolated from Brazilian water buffalo mozzarella cheese

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aminas biogênicas e polifenóis no leite e queijo de ovelhas da raça Bergamácia suplementadas com óleo ou farelo de linhaça (Linum usitassimum L.)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização de segurança e tecnológica de bactérias acidoláticas termofílicas autóctones e aplicação em queijo parmesão

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo peptídico e determinação do perfil de metabólitos de venenos de escorpiões da família buthidae: Tityus serrulatus, Tityus bahiensis e Tityus obscurus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Food safety in wine: optimization of analytical controls and evaluation of production technologies

This PhD thesis has been proposed to validate and then apply innovative analytical methodologies for the determination of compounds with harmful impact on human health, such as biogenic amines and ochratoxin A in wines. Therefore, the influence of production technology (pH, amino acids precursor and use of different malolactic starters) on biogenic amines content in wines was evaluated. An HPLC method for simultaneous determination of amino acids and amines with precolumnderivatization with 9-Fluorenyl-methoxycarbonyl chloride (FMOC-Cl) and UV detection was developed. Initially, the influence of pH, time of derivatization, gradient profile were studied. In order to improve the separation of amino acids and amines and reduce the time of analysis, it was decided to study the influence of different flows and the use of different columns in the chromatographic method. Firstly, a C18 Luna column was used and later two monolithic columns Chromolith in series. It appeared to be suitable for an easy, precise and accurate determination of a relatively large number of amino acids and amines in wines. This method was then applied on different wines produced in the Emilia Romagna region. The investigation permitted to discriminate between red and white wines. Amino acids content is related to the winemaking process. Biogenic amines content in these wines does not represent a possible toxicological problem for human health. The results of the study of influence of technologies and wine composition demonstrated that pH of wines and amino acids content are the most important factors. Particularly wines with pH > 3,5 show higher concentration of biogenic amines than wines with lower pH. The enrichment of wines by nutrients also influences the content of some biogenic amines that are higher in wines added with amino acids precursors. In this study, amino acids and biogenic amines are not statistically affected by strain of lactic acid bacteria inoculated as a starter for malolactic fermentation. An evaluation of different clean-up (SPE-MycoSep; IACs and LLE) and determination methods (HPLC and ELISA) of ochratoxin A was carried out. The results obtained proved that the SPE clean-up are reliable at the same level while the LLE procedures shows lowest recovery. The ELISA method gave a lower determination and a low reproducibility than HPLC method.

Trasduzione del segnale e poliamine nell'apoptosi di cellule cardiache

Introduction: Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy and heart failure, thus representing a potential therapeutic target. Apoptosis of cardiac cells can be induced experimentally by several stimuli including hypoxia, serum withdrawal or combination of both. Several lines of research suggest that neurohormonal mechanisms play a central role in the progression of heart failure. In particular, excessive activation of the sympathetic nervous system or the renin-angiotensin-aldosterone system is known to have deleterious effects on the heart. Recent studies report that norepinephrine (NE), the primary transmitter of sympathetic nervous system, and aldosterone (ALD), which is actively produced in failing human heart, are able to induce apoptosis of rat cardiomyocytes. Polyamines are biogenic amines involved in many cellular processes, including apoptosis. Actually it appears that these molecules can act as promoting, modulating or protective agents in apoptosis depending on apoptotic stimulus and cellular model. We have studied the involvement of polyamines in the apoptosis of cardiac cells induced in a model of simulated ischemia and following treatment with NE or ALD. Methods: H9c2 cardiomyoblasts were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation. Cardiomyocyte cultures were prepared from 1-3 day-old neonatal Wistar rat hearts. Polyamine depletion was obtained by culturing the cells in the presence of α-difluoromethylornithine (DFMO). Polyamines were separated and quantified in acidic cellular extracts by HPLC after derivatization with dansyl chloride. Caspase activity was measured by the cleavage of the fluorogenic peptide substrate. Ornithine decarboxylase (ODC) activity was measured by estimation of the release of 14C-CO2 from 14C-ornithine. DNA fragmentation was visualized by the method of terminal transferase-mediated dUTP nick end-labeling (TUNEL), and DNA laddering on agarose gel electophoresis. Cytochrome c was detected by immunoflorescent staining. Activation of signal transduction pathways was investigated by western blotting. Results: The results indicate that simulated ischemia, NE and ALD cause an early induction of the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, followed by a later increase of caspase activity, a family of proteases that execute the death program and induce cell death. This effect was prevented in the presence of DFMO, an irreversible inhibitor of ODC, thus suggesting that polyamines are involved in the execution of the death program activated by these stimuli. In H9c2 cells DFMO inhibits several molecular events related to apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, down-regulation of Bcl-xL, and DNA fragmentation. The anti-apoptotic protein survivin is down-regulated after ALD or NE treatement and polyamine depletion obtained by DFMO partially opposes survivin decrease. Moreover, a study of key signal transduction pathways governing cell death and survival, revealed an involvement of AMP activated protein kinase (AMPK) and AKT kinase, in the modulation by polyamines of the response of cardiomyocytes to NE. In fact polyamine depleted cells show an altered pattern of AMPK and AKT activation that may contrast apoptosis and appears to result from a differential effect on the specific phosphatases that dephosphorylate and switch off these signaling proteins. Conclusions: These results indicate that polyamines are involved in the execution of the death program activated in cardiac cells by heart failure-related stimuli, like ischemia, ALD and NE, and suggest that their apoptosis facilitating action is mediated by a network of specific phosphatases and kinases.

Studio della maturazione di formaggi pecorino stagionati in stabilimento e in grotta

Aims: Ripening evaluation of two different Pecorino cheese varieties ripened according either to a traditional method in plant and in cave. Different ripening features have been analyzed in order to evaluate the cave as possible ripening environment with the aim of obtaining a peculiar product which could also establish an added value to the cultural heritage of the local place in which it has been originally manufactured. Methods and Results: Chemical-physical features of Pecorino cheese have been initially analyzed into two different ripening environments and experimentations, among which: pH, weight reduction and subsequent water activity. Furthermore, the microbial composition has been characterized in relationship with the two different ripening environments, undertaking a variety of microbial groups, such as: lactic bacteria, staphylococci, yeasts, lactococci, enterobacteria, enterococci. Besides, an additional analysis for the in-cave adaptability evaluation has been the identification of biogenic amines inside the Pecorino cheese (2-phenilethylamine, putrescine, cadaverine, hystidine, tyramine, spermine and spermidine). Further analysis were undertaken in order to track the lipid profile evolution, reporting the concentration of the cheese free fatty acids in object, in relation with ripening time, environment and production. In order to analyse the flavour compounds present in Pecorino cheese, the SPME-GC-MS technique has been widely employed. As a result, it is confirmed the trend showed by the short-chain free fatty acids, that is to say the fatty acids which are mostly involved in conveying a stronger flavor to the cheese. With the purpose of assessing the protheolytic patterns of the above-mentioned Pecorino cheese in the two different ripening environments and testing methods, the technique SDS-PAGE has been employed into the cheese insoluble fraction, whereas the SDS-PAGE technique has been carried out into the cheese soluble portion. Furthermore, different isolated belonging to various microbial groups have been genotypically characterized though the ITS-PCR technique with the aim to identify the membership species. With reference to lactic bacillus the characterized species are: Lactobacillus brevis, Lactobacillus curvatus and Lactobacillus paraplantarum. With reference to lactococci the predominant species is Lactococcus lactis, coming from the employed starter used in the cheese manufacturing. With reference to enterococcus, the predominant species are Enterococcus faecium and Enterococcus faecalis. Moreover, Streptococcus termophilus and Streptococcus macedonicus have been identified too. For staphylococci the identified species are Staphyilococcus equorum, Staphylococcus saprophyfiticus and Staphylococcus xylosus. Finally, a sensorial analysis has been undertaken through on one side a consumer test made by inexperienced consumers, and on the other side through a panel test achieved by expert consumers. From such test Pecorino cheese ripened in cave were found to be more pleasant in comparison with Pecorino cheese ripened in plant. Conclusions: The proposed approach and the undertaken analysis showed the cave as preferential ripening environment for Pecorino cheese and for the development of a more palatable product and safer for consumers’ health.

Identifizierung biogene Amine bildender Bakterien und Einsatz von Enzymen zur Hemmung ihres Wachstums während der Weinbereitung

Mikroorganismen spielen eine wichtige Rolle in der Weinherstellung. Neben ihren positiven Stoffwechselaktivitäten wie die Bildung von Ethanol während der alkoholischen Gärung sind vor allem Bakterien in der Lage, Weinfehler zu verursachen. Einer dieser Weinfehler ist die Produktion von biogenen Aminen. Diese niedermolekularen Stickstoffverbindungen können zu verschiedenen Gesundheitsproblemen wie Bluthochdruck und Migräne führen. Aufgrund von hohen Ethanolgehalten und dem Vorkommen verschiedener biogener Amine kommt es im Wein zu einer Verstärkung dieser physiologischen Effekte. Um die Bildung dieser Verbindungen zu verhindern, ist es von speziellem Interesse, die verantwortlichen Mikroorganismen zu identifizieren und sie in ihrem Wachstum zu hemmen.In einem Teil der Dissertation stand die Isolierung und Identifizierung biogener Amine produzierender Bakterien aus deutschen Jungweinen und Mosten im Vordergrund. Es konnte gezeigt werden, dass hauptsächlich Milchsäurebakterien als potenzielle Produzenten in Frage kommen. Diese Bakteriengruppe war in hohen Titern in nahezu allen Proben vorhanden und stellt somit eine potentielle Gefahr für die Weinbereitung dar. Zur Identifizierung der Isolate wurden verschiedene molekularbiologische Methoden wie specifically amplified DNA polymorphic-PCR (Fingerprintmethode), Multiplex-PCR oder 16S rDNA-Sequenzierung angewandt. Das Screening bezüglich der Bildung von biogenen Aminen erfolgte mit Hilfe einer im Rahmen dieser Arbeit entwickelten hochauflösenden Dünnschichtchromatographie gefolgt von der Quantifizierung mittels HPLC.Zur Wachstumshemmung dieser Schadbakterien wurden zwei Exoenzyme aus Streptomyces albidoflavus B578 isolieren. Diese Enzyme wurden gereinigt und als eine Muramidase und eine Protease identifiziert. Aktivitätstests konnten zeigen, dass diese Enzyme eine hohe lytische Wirkung gegen weinrelevante Mikroorganismen aufweisen. Ebenso war die Aktivität der Enzyme unter Weinbedingungen sehr stabil. Aufgrund dieser Ergebnisse könnten diese Enzyme eine mögliche Alternative zur Zugabe von Lysozym oder Schwefeldioxid sein, welche konventionell in der Weinbereitung ihren Einsatz finden.

[Urinary hormone analysis]

Urinary hormone analysis is applied to detect an altered steroid hormone metabolism, an elevated production of biogenic amines and to non-invasively determine the protein hormone human beta-choriogonadotropin indicating a pregnancy. Occasionally, these determinations need to be complemented by plasma- or serum hormone analysis. Clinical data including current drug therapy and urinary creatinine as reference are required to interpret any urine analysis. Diseases to be investigated by steroid hormone analysis are excess production of a typical or atypical mineralocorticoid active steroid hormones, the hormonal activity of adrenal or ovarian tumors, acne of unknown origin, hirsutism, a PCO-, an adrenogenital or a suspected Cushing syndrome. Biogenic amines should be determined in suspected secondary or refractory arterial hypertension, in case of pheochromocytoma- or paraganglioma-associated symptoms or if a serotonin-producing tumor is suspected. In children genetically determined diseases are the primary background to perform an analysis.

Combine use of Selected Schizosaccharomyces pombe and Lachancea thermotolerans Yeast Strains as an Alternative to the Traditional Malolactic Fermentation in Red Wine Production

Most red wines commercialized in the market use the malolactic fermentationprocess in order to ensure stability from a microbiological point of view. In this secondfermentation, malic acid is converted into L-lactic acid under controlled setups. Howeverthis process is not free from possible collateral effects that on some occasions produceoff-flavors, wine quality loss and human health problems. In warm viticulture regions suchas the south of Spain, the risk of suffering a deviation during the malolactic fermentationprocess increases due to the high must pH. This contributes to produce wines with highvolatile acidity and biogenic amine values. This manuscript develops a new red winemakingmethodology that consists of combining the use of two non-Saccharomyces yeast strains asan alternative to the traditional malolactic fermentation. In this method, malic acid is totallyconsumed by Schizosaccharomyces pombe, thus achieving the microbiological stabilizationobjective, while Lachancea thermotolerans produces lactic acid in order not to reduce andeven increase the acidity of wines produced from low acidity musts. This technique reducesthe risks inherent to the malolactic fermentation process when performed in warm regions.The result is more fruity wines that contain less acetic acid and biogenic amines than thetraditional controls that have undergone the classical malolactic fermentation.

Patch-clamp and amperometric recordings from norepinephrine transporters: Channel activity and voltage-dependent uptake

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants, cocaine, and amphetamines. Despite their prominent role in neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.

Perfil conductual de las drogas de diseño MBDB, MDEA y PMA en modelos animales de agresión y ansiedad

MBDB, MDEA y PMA son tres drogas de diseño, estructuralmente similares al MDMA (“éxtasis”), que se han identificado en la composición de pastillas distribuidas como “éxtasis” en entornos recreativos durante los últimos treinta años. Estas feniletilaminas sintéticas presentan un perfil psicotrópico de tipo entactógeno (con capacidad para facilitar la proximidad, el contacto y la comunicación empática), similar al del MDMA en el modelo de discriminación de drogas. El MDMA ha sido objeto de un creciente interés científico y es, hasta la fecha, la única sustancia con un perfil entactógeno de la que se han investigado sus efectos conductuales en modelos animales de agresión y ansiedad, si bien sus resultados no siempre coinciden. Aunque existen algunas evidencias de que el MDMA puede tener efectos ansiolíticos en animales de laboratorio (Lin, Burden, Christie, & Johnston, 1999; Morley & McGregor, 2000; Ho, Pawlak, Guo, & Schwarting, 2004), en otros estudios se han observado alteraciones conductuales y correlatos neuroquímicos que sugieren un efecto ansiogénico (Bhattacharya, Bhattacharya & Ghosal, 1998; Gurtman, Morley, Li, Hunt, & McGregor, 2002; Maldonado & Navarro, 2000;; Navarro & Maldonado, 2002). Asimismo, en otros trabajos se ha señalado que el MDMA induce efectos antiagresivos (reducción de las conductas de amenaza y ataque), que se acompañan de un marcado aumento de las conductas de evitación/huida y defensa/sumisión, así como de una reducción de las conductas de investigación social, sugiriendo también la existencia de un perfil ansiogénico en los encuentros agonísticos entre ratones machos (Maldonado & Navarro, 2001; Navarro & Maldonado, 1999). En contraste, hasta la fecha la información experimental de las drogas MBDB, MDEA y PMA se limita a la evaluación de sus efectos conductuales sobre la conducta motora, así como algunos estudios sobre su metabolismo y posible mecanismo de acción. El objetivo general de este trabajo de investigación ha sido estudiar el perfil conductual de MBDB, MDEA y PMA en modelos animales de agresión y ansiedad. Para ello, se han examinado los efectos del MBDB (2, 4 y 8 mg/kg), MDEA (5, 10 y 20 mg/kg) y PMA (2, 4, 8 y 12 mg/kg) utilizando el modelo de agresión inducida por aislamiento y el modelo de ansiedad del laberinto elevado en cruz en ratones machos. Los resultados indican que estas sustancias en general comparten un perfil antiagresivo inespecífico. Esta falta de especificidad se debe en unos casos al aumento de las conductas de inmovilidad (4-12 mg/kg PMA), pero también a la presencia de propiedades ansiogénicas durante la interacción social, en especial con dosis elevadas, mientras que solo las dosis más bajas parecen aumentar la proximidad social, en especial la dosis menor de MBDB. Además, estas drogas parecen alterar el patrón conductual agonístico ofensivo (MBDB y MDEA) y defensivo (MBDB, MDEA y PMA), produciendo cambios diádicos que resultan coherentes con un aumento del nivel de conflicto y de ansiedad. En consonancia, los resultados del modelo del laberinto elevado en cruz indican que el MBDB produce un aumento de la ansiedad de menor intensidad que el producido por el MDMA. Sin embargo, MDEA y PMA parecen generar un estado de hipoexploración, y solo en dosis determinadas (20 mg/kg de MDEA y 4 mg/kg de PMA) muestran alteraciones discretas que sugieren un efecto ansiogénico débil, un perfil que en conjunto podría sugerir cierta similitud con alteraciones conductuales propias de los compuestos alucinógenos. Debido a la diferencia del perfil conductual del MDEA y PMA hallados en ambos modelos, sería necesario evaluar la ansiedad y su posible relación con la dosis y/o con la presencia de un oponente en la prueba en otros modelos experimentales. Lin, H. Q., Burden, P. M., Christie, M. J., & Johnston, G. A. R. (1999). The anxiogenic-like and anxiolytic-like effects of MDMA on mice in the elevated plus-maze: A comparison with amphetamine. Pharmacology, Biochemistry and Behavior, 62(3), 403-408. Morley, K. C., & McGregor, I. S. (2000). (±)-3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') increases social interaction in rats. European Journal of Pharmacology, 408(1), 41-49. Bhattacharya, S. K., Bhattacharya, A., & Ghosal, S. (1998). Anxiogenic activity of methylenedioxymethamphetamine (Ecstasy): An experimental study. Biogenic Amines, 14(3), 217-237. Gurtman, C. G., Morley, K. C., Li, K. M., Hunt, G. E., & McGregor, I. S. (2002). Increased anxiety in rats after 3,4-methylenedioxymethamphetamine: Association with serotonin depletion. European Journal of Pharmacology, 446(1-3), 89-96. Ho, Y., Pawlak, C. R., Guo, L., & Schwarting, R. K. W. (2004). Acute and long-term consequences of single MDMA administration in relation to individual anxiety levels in the rat. Behavioural Brain Research, 149(2), 135-144. Maldonado, E., & Navarro, J. F. (2000). Effects of 3,4-methylenedioxy-methamphetamine (MDMA) on anxiety in mice tested in the light/dark box. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 24(3), 463-472. Maldonado, E., & Navarro, J. F. (2001b). MDMA ('ecstasy') exhibits an anxiogenic-like activity in social encounters between male mice. Pharmacological Research, 44(1), 27-31. Navarro, J. F., & Maldonado, E. (1999). Behavioral profile of 3,4-methylenedioxy-methamphetamine (MDMA) in agonistic encounters between male mice. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 23(2), 327-334. Navarro, J. F., & Maldonado, E. (2002). Acute and subchronic effects of MDMA ("ecstasy") on anxiety in male mice tested in the elevated plus-maze. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 26(6), 1151-1154.

Characterisation of "Catalão" and "Salsichão" Portuguese traditional sausages with salt reduction

The present study evaluated the effect of salt reduction on traditional dry-cured sausages' safety, quality and product acceptance, comprising physicochemical and microbiological parameters, biogenic amines, fatty acids, texture profile and sensory analysis. According to our results, salt content had a major effect on microbiological counts, although not compromising the products' safety. Marked differences were identified regarding biogenic amines, in particular for histamine, tyramine and cadaverine, which were detected in larger amounts in products with 3%. Moreover, significant differences in the fatty acids profile have also been found, but only in less abundant components such as linoleic, lauric and heneicosanoic acids. Texture profile analysis of low-salt products, revealed a decrease in hardness and chewiness, along with an increase in adhesiveness values. Sensory evaluations revealed that despite the less intense aroma, products with 3% salt, had a more balanced salt perception. Our results suggest that salt content may be reduced to 50% in dry-cured products, with the obvious health-related advantages.