Die Aktivierung der Speichelsekretion erfolgt in der innervierten Speicheldrüse der Schabe <i>Periplaneta americana</i> durch die biogenen Amine Dopamin (DA) und Serotonin (5-HT). Die Acini der Speicheldrüse sezernieren einen Primärspeichel, der in den Ausführgängen modifiziert wird. Die durch DA und 5-HT aktivierten Signalwege sowie die an der Elektrolyt- und Flüssigkeitssekretion bzw. Speichel-modifikation beteiligten Transportmechanismen sind weitgehend unbekannt.<br> Mikrofluorometrische Ca²⁺-, Na⁺- und pH-Messungen in Kombination mit pharmakologischen Experimenten, biochemische Messungen der Aktivitäten von Ionentransport-ATPasen sowie videomikroskopische Analysen zu transepithelialen Wasserbewegungen wurden in dieser Arbeit durchgeführt. Sie sollten Informationen über die an der Speichelbildung und -modifikation beteiligten Transportmechanismen und die Signalwege liefern, welche durch DA und/oder 5-HT aktiviert werden. <br><br> Wesentliche Ergebnisse dieser Arbeit waren:<br><br> <ul> <li>Messungen des intrazellulären pH (pH_i) in Gangzellen zeigten, dass isolierte Ausführgänge mit Acini bei Stimulierung mit DA und 5-HT stark ansäuerten. In isolierten Ausführgängen ohne Acini verursachte nur DA eine schwache Ansäuerung. Da nur die Ausführgänge dopaminerg innerviert sind, die Acini jedoch dopaminerg und serotonerg, zeigt dieses Ergebnis, dass die DA- und/oder 5-HT-induzierte Primärspeichelbildung die Ursache für die pHi-Änderungen in den Gangzellen ist. pH_i-Messungen in den Gangzellen geben also auch Hinweise auf Transportvorgänge in den Acini.</li> <li> Der Na⁺-K⁺-2Cl^--Symporter und der Cl^--HCO₃^--Antiporter, gekoppelt mit dem Na⁺ H⁺-Antiporter (NHE) waren an der NaCl-Aufnahme in die peripheren Zellen der Acini zur Bildung des NaCl-reichen Primärspeichels beteiligt. Die Aktivität dieser Transporter hing von der CO₂/HCO₃^--Verfügbarkeit ab und war Ca²⁺-abhängig.</li> <li>Die starke Ansäuerung in den Gangzellen hing nicht von der Aktivität der apikalen vakuolären Protonen-ATPase (V-H⁺-ATPase), aber von der Aktivität der basolateralen Na⁺-K⁺-ATPase ab, die anscheinend in den Ausführgängen die Speichelmodifikation energetisiert.</li> <li>In isolierten Ausführgängen mit Acini waren die V-H⁺-ATPase und Na⁺-abhängige Transporter (u. a. NHE) an der Erholung von einer DA-induzierten oder einer NH₄Cl-Vorpuls-induzierten Ansäuerung in den Gangzellen beteiligt. Bei der Regulation des pH_i in unstimulierten Gangzellen spielten diese Transporter keine Rolle.</li> <li>In isolierten Ausführgängen mit Acini induzierte DA in den Gangzellen einen Anstieg der [Na⁺]_i und, zeitlich verzögert, auch der [Ca²⁺]_i. Der [Na⁺]_i-Anstieg war von der Aktivität der Acini abhängig und erfolgte möglicherweise über apikale Na+-Kanäle. Der [Ca²⁺]_i-Anstieg war graduiert und tonisch. Der DA-induzierte [Na⁺]_i-Anstieg in den Gangzellen und deren Depolarisation führten dazu, dass der basolaterale Na⁺-Ca²⁺-Antiporter in den Ca²⁺-Influx-Modus umkehrte. Die daraus resultierende tonische [Ca²⁺]_i-Erhöhung könnte an der Regulation der Na⁺-Rückresorption beteiligt sein.</li> <li>Zum Nachweis transepithelialer Flüssigkeitsbewegungen in isolierten Ausführgängen wurde eine videomikroskopische Methode entwickelt. Isolierte Ausführgänge ohne Acini resorbierten im unstimulierten Zustand Flüssigkeit aus dem Ausführganglumen. Möglicherweise sezernieren die Acini auch im unstimulierten Zustand mit geringerer Rate einen Primärspeichel, der in den Ausführgängen resorbiert wird. Die Resorption war ATP-abhängig. Der ATP-verbrauchende Transportmechanismus konnte nicht identifiziert werden. Weder die Na⁺-K⁺-ATPase noch die V-H⁺-ATPase waren an der Resorption beteiligt.</li> </ul> <br> Diese Arbeit trug zur Kenntnis der komplexen Funktionsweise von Speicheldrüsen in Insekten bei und erweiterte das lückenhafte Wissen über die zellulären Wirkungen biogener Amine in Insekten. Zudem wurden in dieser Arbeit viele Parallelen zu Funktionsweisen der Speicheldrüsen in Vertebraten deutlich. / The acinar salivary glands in the cockroach <i>Periplaneta americana</i> are innervated by dopaminergic and serotonergic fibers and secrete a NaCl-rich primary saliva upon stimulation with the biogenic amines dopamine (DA) or serotonin (5-HT). The ducts downstream of the acini are thought to modify the primary saliva by Na⁺reabsorption and K⁺ secretion. The electrolyte and fluid transport processes activated by DA and 5-HT as well as the second messenger pathways mediating between the biogenic amine receptors and the effector transport mechanisms are poorly understood.In this sudy, microfluorometrical Ca²⁺, Na⁺ and pH measurements were performed in combination with pharmacological experiments. Furthermore, ATPase activity assays and microscopical analyses of transepithelial fluid transport were done. The aim of this work has been the characterisation of the DA-induced transport mechanisms in the cockroach salivary glands in order to improve our understanding of the cellular actions of biogenic amines in insects. <br><br> Intracellular pH measurements in duct cells of isolated small lobes of salivary glands consiting of several acini and ducts showed a strong intracellular acidification upon DA or 5-HT stimulation. On the other hand, only a small intracellular acidification could be recognised in isolated ducts without acini. The acini are innervated by dopaminergic and serotonergic fibers, whereas the ducts are innervated only by dopaminergic fibers. Thus, this result demonstrates, that the DA- or 5-HT-induced production of primary saliva in the acini causes the intracellular pH changes in the ducts. Consequently, intracellular pH measurements in ducts are also useful to characterise transport processes in the acini.<br><br> The Na⁺-K⁺-2Cl^- cotransport and/or the Cl^--HCO₃^- exchange combined with the Na⁺ H⁺ exchange (NHE) were responsible for the NaCl uptake at the basolateral membrane in the peripheral cells of the acini during production of primary saliva. The activity of these transporters was regulated by the CO₂/HCO₃^--availability and was Ca²⁺-dependent. The activity of the basolateral Na⁺-K⁺-ATPase, but not of the apical vacuolar-type proton pump (V-H⁺-ATPase) in the duct cells was necessary for the strong intracellular acidification in the ducts with acini. Thus, the Na⁺-K⁺-ATPase seems to energise the saliva modification in the ducts. In ducts with acini, the V-H⁺-ATPase and Na⁺-dependent transporters (e.g. NHE) were responsible for the pH-recovery after a DA- or NH₄Cl-induced intracellular acidification in the duct cells. In the regulation of the intracellular resting pH these transporters played a minor role. In addition, DA induced an increase in the intracellular Na⁺ concentration, followed by an increase in the intracellular Ca²⁺ concentration in duct cells with acini, but never in duct cells without acini. The Na⁺ elevation was probably the result of the activity of apical Na⁺ channels. The DA-induced Na⁺ elevation and a depolarisation of the basolateral membrane of the duct cells reversed a Na⁺-Ca²⁺ exchange activity into the reverse mode causing a graded Ca²⁺ elevation in duct cells. The Ca²⁺ elevation is probably involved in the regulation of the Na⁺ reabsorption during saliva modification. Transepithelial fluid transport in isolated ducts was detected with a fluorescent microscopical method. Already unstimulated isolated ducts reabsorbed fluid from the duct lumen to the bath side. Perhaps unstimulated acini possess a basic secretion rate and this primary saliva is than reabsorbed in the ducts. The fluid reabsorption was ATP-dependent, but the ATP-consuming transport mechanism could not be identified. Neither the basolateral Na⁺-K⁺-ATPase, nor the apical V-H⁺-ATPase were involved in fluid reabsorption. This work extends our knowledge about the complex function of insect salivary glands and about the cellular action of biogenic amines in insects. Additionally, it indicates lots of similarities between the functions of salivary glands in vertebrates and invertebrates.

Biogene Amine sind kleine organische Verbindungen, die sowohl bei Wirbeltieren als auch bei Wirbellosen als Neurotransmitter, Neuromodulatoren und/oder Neurohormone wirken können. Sie bilden eine bedeutende Gruppe von Botenstoffen und entfalten ihre Wirkungen über die Bindung an eine bestimmte Klasse von Rezeptorproteinen, die als G-Protein-gekoppelte Rezeptoren bezeichnet werden. Bei Insekten gehören zur Substanzklasse der biogenen Amine die Botenstoffe Dopamin, Tyramin, Octopamin, Serotonin und Histamin. Neben vielen anderen Wirkung ist z.B. gezeigt worden, daß einige dieser biogenen Amine bei der Honigbiene (Apis mellifera) die Geschmacksempfindlichkeit für Zuckerwasser-Reize modulieren können. Ich habe verschiedene Aspekte der aminergen Signaltransduktion an den „Modellorganismen“ Honigbiene und Amerikanische Großschabe (Periplaneta americana) untersucht. Aus der Honigbiene, einem „Modellorganismus“ für das Studium von Lern- und Gedächtnisvorgängen, wurden zwei Dopamin-Rezeptoren, ein Tyramin-Rezeptor, ein Octopamin-Rezeptor und ein Serotonin-Rezeptor charakterisiert. Die Rezeptoren wurden in kultivierten Säugerzellen exprimiert, um ihre pharmakologischen und funktionellen Eigenschaften (Kopplung an intrazelluläre Botenstoffwege) zu analysieren. Weiterhin wurde mit Hilfe verschiedener Techniken (RT-PCR, Northern-Blotting, in situ-Hybridisierung) untersucht, wo und wann während der Entwicklung die entsprechenden Rezeptor-mRNAs im Gehirn der Honigbiene exprimiert werden. Als Modellobjekt zur Untersuchung der zellulären Wirkungen biogener Amine wurden die Speicheldrüsen der Amerikanischen Großschabe genutzt. An isolierten Speicheldrüsen läßt sich sowohl mit Dopamin als auch mit Serotonin Speichelproduktion auslösen, wobei Speichelarten unterschiedlicher Zusammensetzung gebildet werden. Dopamin induziert die Bildung eines völlig proteinfreien, wäßrigen Speichels. Serotonin bewirkt die Sekretion eines proteinhaltigen Speichels. Die Serotonin-induzierte Proteinsekretion wird durch eine Erhöhung der Konzentration des intrazellulären Botenstoffs cAMP vermittelt. Es wurden die pharmakologischen Eigenschaften der Dopamin-Rezeptoren der Schaben-Speicheldrüsen untersucht sowie mit der molekularen Charakterisierung putativer aminerger Rezeptoren der Schabe begonnen. Weiterhin habe ich das ebony-Gen der Schabe charakterisiert. Dieses Gen kodiert für ein Enzym, das wahrscheinlich bei der Schabe (wie bei anderen Insekten) an der Inaktivierung biogener Amine beteiligt ist und im Gehirn und in den Speicheldrüsen der Schabe exprimiert wird. / Biogenic amines are small organic compounds that act as neurotransmitters, neuromodulators and/or neurohormones in vertebrates and in invertebrates. They form an important group of messenger substances and mediate their diverse effects by binding to membrane receptors that primarily belong to the large gene-family of G protein-coupled receptors. In insects, the group of biogenic amine messengers consists of five members: dopamine, tyramine, octopamine, serotonin, and histamine. Besides many other effects, some of these biogenic amines were shown, for example, to modulate gustatory sensitivity to sucrose stimuli in the honeybee (Apis mellifera). I have investigated various aspects of the aminergic signal transduction in the “model organisms” honeybee and American cockroach (Periplaneta americana). So far, I have characterized two dopamine receptors, a tyramine receptor, an octopamine receptor and a serotonin receptor of the honeybee, which is well-known for its learning and memory capacities. The receptors where expressed in cultivated mammalian cells in order to analyze their pharmacological and functional (i.e., second messenger coupling) properties. The spatiotemporal expression patterns of the respective receptor mRNA were investigated in the honeybee brain by using different techniques (RT PCR, Northern blotting, in situ-hybridization). The salivary glands of the American cockroach were used as a model object in order to investigate the cellular effects of biogenic amines. Both dopamine and serotonin trigger salivary secretion in isolated salivary glands. The quality of the secreted saliva is, however, different. Stimulation of the glands by serotonin results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Serotonin-induced protein secretion is mediated by an increase in the intracellular concentration of cAMP. The pharmacological properties of dopamine receptors associated with cockroach salivary glands were investigated and the molecular characterization of putative aminergic receptors of the cockroach was initiated. Furthermore, I have characterized the ebony gene of the cockroach. This gene encodes an enzyme that is probably involved in the inactivation of biogenic amines in the cockroach (as in other insects). The ebony gene is expressed in the brain and in the salivary glands of the cockroach.

L’ocratossina e le ammine biogene sono due metaboliti biologici che possono essere ritrovati nei vini. Il primo di questi contaminanti è stato studiato recentemente per la sua elevata tossicità sebbene non sia rintracciabile frequentemente nei vini. Le ammine biogene sono presenti in ogni tipo di vino in differenti concentrazioni, oltretutto, alcune di loro, ad alte concentrazioni, possono causare reazioni allergiche. Gli obiettivi di questo PhD riguardano tre differenti aspetti. Indagare metodi biologici di per la decontaminazione di ocratossina A durante la vinificazione; in particolare lo studio si è focalizzato sull’uso di un ceppo Lactobacillus plantarum utilizzato come starter malolattico. Valutare la presenza di Lactobacillus spp., isolati da mosto e vino, in grado di produrre ammine biogene, usando tecniche molecolari come la reazione a catena della polimerasi (PCR) per rilevare i geni codificanti gli enzimi responsabili della sintesi di questi composti. Testare la capacità di un L. plantarum di effettuare la malolattica effettuando l’inoculo in differenti fasi della vinificazione e valutare il trend delle ammine biogene già presenti nel mosto. / Two of the major biological metabolites present in wine are the ochratoxin and the biogenic amines. The first of these contaminants was studied in recent decades because of its toxicity in humans, although its presence is not frequent in wines. The biogenic amines are present in every types of wine in different concentration, and some of them, in high concentrations, can cause allergenic reactions in humans. The objectives of this PhD regard three different aspects. Investigate the biological methods to reduce ochratoxin A in wine during winemaking; in particular the study is focused to use a Lactobacillus plantarum strain as malolactic starter. Investigate the presence of Lactobacillus spp., isolated from must and wine, able to produce the amines, using molecular techniques as polymerase chain reaction (PCR) to detect the genes that encode for the enzymes responsible of the synthesis of these compounds. Test the ability of a L. plantarum to perform MLF in relationship with inoculation time and assess the trend of biogenic amines already present in must.

Gram-positive bacteria are part of the normal flora of fish from different aquatic environments. They are mesophilic bacteria and demonstrate optimum growth at ambient temperature. In the sub-tropics, marine fish are caught from seas at temperatures of 16 to 34C, they are usually not iced and are handled at ambient temperature. It was hypothesized that under these conditions Gram-positive bacteria will be abundant in sub-tropical marine fish and will have roles in the spoilage of fish. A review of literature showed that there is a gap in understanding the Gram-positive bacterial populations in sub-tropical marine fish. This is partly due to the fact that the selective media used for isolating Gram-positive bacteria have limitations. Ecological and speciation studies have revealed that the ecology and speciation of many Gram-positive bacteria have not been clearly elucidated. The effect of ambient storage on the individual genera and species of Gram-positive bacteria in fish has been rarely studied. The spoilage potential of Gram-positive bacteria of marine fish origin has not been clearly determined. Therefore, the main aims of this study were to isolate Gram-positive bacteria from fresh and ambient-temperature-stored sub-tropical marine fish, speciate the isolates and study the spoilage potential of the isolates. The practical components of this study were conducted in four parts. The first part dealt with validation of tryptone soya agar with 0.25% phenylethyl alcohol (PEA-TSA) to enumerate Gram-positive bacteria. The second part enumerated Gram-positive bacteria from the muscles, gills and gut of Pseudocaranx dentex (Silver Trevally), Pagrus auratus (Snapper) and Mugil cephalus (Sea Mullet) stored at 25C for 15 hours using PEA-TSA. The third part dealt with the speciation of the isolates using appropriate methods such as polymerase chain reaction, 16S rRNA gene sequence, the VITEK JR system and conventional biochemical methods. In the fourth part, the isolates were assayed qualitatively for their ability to produce volatile sulphur compounds (VSC), reduce trimethylamine oxide and decarboxylate histidine, lysine and ornithine at mesophilic temperature, 32C. Initial studies indicated that PEA-TSA significantly (P< 0.05) reduced the total aerobic bacterial count of fish whereas control Gram-positive bacteria were not affected (P> 0.05). Gram-positive aerobic bacterial counts (GABC) significantly (P< 0.05) increased in the muscles and gills during ambient storage for 15 hours. Within each species, no significant (P> 0.05) differences were found in GABC between muscles and gills. Moreover, there were no significant differences (P> 0.05) in GABC between fish species during storage. In total, 390 bacteria were isolated from the fresh and stored fish; 339 isolates (87%) were found to be Gram-positive. Two hundred and sixty-six isolates (78%) of Gram-positive bacteria were identified to fall into 13 genera, namely Staphylococcus, Micrococcus, Bacillus, Virgibacillus, Brevibacillus, Corynebacterium, Streptococcus, Enterococcus, Aerococcus, Exiguobacterium, Carnobacterium, Vagococcus and Sporosarcina and 30 species. In fresh fish, Staphylococcus epidermidis and Micrococcus luteus were the most frequent isolates. The effect of storage at 25C for 15 hours resulted in a change of Gram-positive bacterial populations; while S. epidermidis, S. xylosus and Bacillus megaterium were no longer present, S. warneri, B. sphaericus, Brevibacillus borstelensis, Enterococcus faecium and Streptococcus uberis increased. Three species, E. faecium, Str. uberis and B. sphaericus, were the most prevalent at the end of storage. Micrococcus luteus and S. warneri were the most prevalent isolates from Pseudocaranx dentex, but E. faecium and Str. uberis were the most frequently isolated from Pagrus auratus and Mugil cephalus. With respect to different parts of the fish body, E. faecium, Str. uberis and B. sphaericus were the most frequent isolates from the muscles, E. faecium, Str. uberis from the gills and M. luteus from the gut. Among the 228 isolates examined, Br. borstelensis 73, Br. borstelensis 291, Str. uberis 339, Vagococcus fluvialis 31 and Vag. fluvialis 132 produced VSC from sodium thiosulphate, cysteine and methionine. However, strains varied in sulphur source utilization. Exiguobacterium acetylicum 5, Exiguobacterium spp. 191, Carnobacterium spp. 338, Br. borstelensis 73, Br. borstelensis 291, Str. uberis 30, Str. uberis 339, Vag. fluvialis 31 and Vag. fluvialis 132 reduced TMAO. No histidine decarboxylase activity was found in the Gram-positive bacterial species tested. Lysine and ornithine were decarboxylated mainly by different strains of S. warneri, S. epidermidis and M. luteus. During ambient storage of fish, the frequency of lysine-decarboxylating bacteria increased and became more diverse after 5 hours of storage. Among fish species examined, the frequencies of lysine- and ornithine-decarboxylating bacteria were higher and more diverse in Pseudocaranx dentex than in Pagrus auratus and Mugil cephalus. This study found that Gram-positive bacteria were abundant and diverse in sub-tropical marine fish; however, their frequencies were affected by fish habitat and fish body part. Ambient temperature storage determined which Gram-positive bacterial species were dominant. With the exception of one isolate of S. aureus, Gram-positive bacteria isolated from sub-tropical marine fish caught from unpolluted water were not potential pathogens. The study also showed that Gram-positive bacteria had greater ability to decarboxylate lysine and ornithine than to produce VSC or reduce TMAO, and the spoilage potential of a bacterial species was a strain-dependent behaviour. This is a significant study as it is the first study on this aspect sub-tropical marine fish. It validated a selective medium that can be used to enumerate most Gram-positive bacteria from a marine environment. Most of the Gram-positive bacterial species from sub-tropical marine fish identified in this study were documented for the first time. The effects of ambient storage and the spoilage potential of Gram-positive bacteria from sub-tropical marine were clearly elucidated.

O gênero Psychotria destaca-se na família RUBIACEAE pela produção de alcalóides bioativos e por sua taxonomia complexa, sendo muitas vezes relacionado aos gêneros Palicourea, Cephaelis, Calycodendron e Calycosia. A divisão de Psychotria nos subgêneros Psychotria, Tetramerae e Heteropsychotria foi proposta com o objetivo de auxiliar a classificação quimiotaxonômica do gênero. Estudos demonstram que o subgênero Psychotria (espécies pantropicais) produz alcalóides poliméricos, formados por duas ou mais unidades de triptamina; enquanto o subgênero Heteropsychotria parece estar envolvido com a produção de alcalóides indol monoterpênicos, de acordo com pesquisas realizadas com diferentes espécies coletadas no Sul do Brasil. O isolamento e purificação dos alcalóides estrictosamida, ácido estrictosidínico e miriantosina, a partir de Psychotria myriantha, corroboram com esta hipótese, permitindo a inclusão da espécie no subgênero Heteropsychotria. A literatura descreve várias atividades para extratos e alcalóides isolados de espécies de Psychotria, como antimicrobiana e analgésica, por exemplo. Neste trabalho, o extrato n-butanólico de alcalóides de P. myriantha, além de seus alcalóides isolados, apresentaram atividade inibidora da migração de leucócitos, sugerindo um efeito antiinflamatório, e capacidade de inibir a ação da enzima acetilcolinesterase. Extratos e alcalóides isolados da espécie foram avaliados quanto à atividade antioxidante em CCD, frente ao DPPH, apresentando resultado negativo. O extrato EBA e o alcalóide ácido estrictosidínico aumentaram o tempo de latência no teste da retirada da cauda frente ao estímulo térmico, indicando uma atividade analgésica do tipo opióide. A influência do ácido estrictosidínico, alcalóide isolado em maior quantidade em massa, sobre os níveis de DA, DOPAC, 3-MT, HVA, 5-HT e 5-HIAA em estruturas cerebrais de ratos foi verificada. Hipocampos de animais que receberam injeção intra-hipocampal bilateral de ácido estrictosidínico (20 μg/μL) apresentaram redução de 83,4 % nos níveis de serotonina, em comparação ao grupo controle, enquanto os córtices desses animais apresentaram redução nos níveis de DOPAC (35,9%), 3-MT (24,7%) e 5-HIAA (9%). Hipocampos e estriados de ratos tratados com injeção i.p. de ácido estrictosidínico (10 mg/kg) demonstraram diminuição de 63,4 e 28,7% nos níveis de 5-HT, respectivamente. As alterações nos níveis de aminas biogênicas nas estruturas avaliadas, além das atividades analgésica e inibidora da acetilcolinesterase, indicam que P. myriantha e espécies do subgênero Heteropsychotria constituem uma potencial fonte de substâncias bioativas no tratamento de distúrbios do sistema nervoso central. / Psychotria genus is an important in RUBIACEAE due to bioactive alkaloids production and complex taxonomy, being related to Palicourea, Cephaelis, Calycodendron and Calycosia genera. The division of Psychotria in Psychotria, Tetramerae and Heteropsychotria subgenera was proposed with the aim of aiding the genus chemotaxonomic classification. Studies demonstrate that Psychotria subgenus produce polyindoline alkaloids formed by two or more triptamine units; whereas Heteropsychotria subgenus seems to be involved with indole monoterpene alkaloids production, according to researches with different species collected in Southern Brazil. Isolation and purification of strictosamide, strictosidinic acid and miriantosine from Psychotria myriantha corroborated this hypothesis, allowing its inclusion into Heteropsychotria subgenus. The scientific literature describes several activities to Psychotria extracts and alkaloids, such as antimicrobial and analgesic, for example. In this work, P. myriantha n-butanolic alkaloid extract and isolated compounds inhibited the leukocyte migration, suggesting an antiinflammatory activity, and a weak ability to inhibit the action of acetylcholinesterase enzyme. Alkaloids and extracts from P. myriantha were evaluated as regards their antioxidant activity using DPPH, with no positive results. EBA and strictosidinic acid increased the latency in the tail flick model, indicating an opioid analgesic activity. The influence of strictosidinic acid on the levels of DA, DOPAC, 3-MT, HVA, 5-HT and 5-HIAA in brain structures of rats was verified. Hippocampus with intra-hippocampal injection of strictosidinic acid (20 μg/μL) displayed a decrease of 83.4% in serotonin levels, in comparison with control group; whereas the cortex showed a decrease in the levels of DOPAC (35.9%), 3-MT (24.7%) and 5-HIAA (9%). Hippocampus and striatum that received intraperitoneal injection of strictosidinic acid (10 mg/kg) showed 5-HT levels reduction of 63.4 e 28.7%, respectively. The biogenic amine levels alterations in the studied structures, associated with the analgesic and acetylcholinesterase inhibitor activities, suggest that P. myriantha and species from the Heteropsychotria subgenus constitute a font of bioactive compounds in the treatment of central nervous system disturbs.

O gênero Psychotria destaca-se na família RUBIACEAE pela produção de alcalóides bioativos e por sua taxonomia complexa, sendo muitas vezes relacionado aos gêneros Palicourea, Cephaelis, Calycodendron e Calycosia. A divisão de Psychotria nos subgêneros Psychotria, Tetramerae e Heteropsychotria foi proposta com o objetivo de auxiliar a classificação quimiotaxonômica do gênero. Estudos demonstram que o subgênero Psychotria (espécies pantropicais) produz alcalóides poliméricos, formados por duas ou mais unidades de triptamina; enquanto o subgênero Heteropsychotria parece estar envolvido com a produção de alcalóides indol monoterpênicos, de acordo com pesquisas realizadas com diferentes espécies coletadas no Sul do Brasil. O isolamento e purificação dos alcalóides estrictosamida, ácido estrictosidínico e miriantosina, a partir de Psychotria myriantha, corroboram com esta hipótese, permitindo a inclusão da espécie no subgênero Heteropsychotria. A literatura descreve várias atividades para extratos e alcalóides isolados de espécies de Psychotria, como antimicrobiana e analgésica, por exemplo. Neste trabalho, o extrato n-butanólico de alcalóides de P. myriantha, além de seus alcalóides isolados, apresentaram atividade inibidora da migração de leucócitos, sugerindo um efeito antiinflamatório, e capacidade de inibir a ação da enzima acetilcolinesterase. Extratos e alcalóides isolados da espécie foram avaliados quanto à atividade antioxidante em CCD, frente ao DPPH, apresentando resultado negativo. O extrato EBA e o alcalóide ácido estrictosidínico aumentaram o tempo de latência no teste da retirada da cauda frente ao estímulo térmico, indicando uma atividade analgésica do tipo opióide. A influência do ácido estrictosidínico, alcalóide isolado em maior quantidade em massa, sobre os níveis de DA, DOPAC, 3-MT, HVA, 5-HT e 5-HIAA em estruturas cerebrais de ratos foi verificada. Hipocampos de animais que receberam injeção intra-hipocampal bilateral de ácido estrictosidínico (20 μg/μL) apresentaram redução de 83,4 % nos níveis de serotonina, em comparação ao grupo controle, enquanto os córtices desses animais apresentaram redução nos níveis de DOPAC (35,9%), 3-MT (24,7%) e 5-HIAA (9%). Hipocampos e estriados de ratos tratados com injeção i.p. de ácido estrictosidínico (10 mg/kg) demonstraram diminuição de 63,4 e 28,7% nos níveis de 5-HT, respectivamente. As alterações nos níveis de aminas biogênicas nas estruturas avaliadas, além das atividades analgésica e inibidora da acetilcolinesterase, indicam que P. myriantha e espécies do subgênero Heteropsychotria constituem uma potencial fonte de substâncias bioativas no tratamento de distúrbios do sistema nervoso central. / Psychotria genus is an important in RUBIACEAE due to bioactive alkaloids production and complex taxonomy, being related to Palicourea, Cephaelis, Calycodendron and Calycosia genera. The division of Psychotria in Psychotria, Tetramerae and Heteropsychotria subgenera was proposed with the aim of aiding the genus chemotaxonomic classification. Studies demonstrate that Psychotria subgenus produce polyindoline alkaloids formed by two or more triptamine units; whereas Heteropsychotria subgenus seems to be involved with indole monoterpene alkaloids production, according to researches with different species collected in Southern Brazil. Isolation and purification of strictosamide, strictosidinic acid and miriantosine from Psychotria myriantha corroborated this hypothesis, allowing its inclusion into Heteropsychotria subgenus. The scientific literature describes several activities to Psychotria extracts and alkaloids, such as antimicrobial and analgesic, for example. In this work, P. myriantha n-butanolic alkaloid extract and isolated compounds inhibited the leukocyte migration, suggesting an antiinflammatory activity, and a weak ability to inhibit the action of acetylcholinesterase enzyme. Alkaloids and extracts from P. myriantha were evaluated as regards their antioxidant activity using DPPH, with no positive results. EBA and strictosidinic acid increased the latency in the tail flick model, indicating an opioid analgesic activity. The influence of strictosidinic acid on the levels of DA, DOPAC, 3-MT, HVA, 5-HT and 5-HIAA in brain structures of rats was verified. Hippocampus with intra-hippocampal injection of strictosidinic acid (20 μg/μL) displayed a decrease of 83.4% in serotonin levels, in comparison with control group; whereas the cortex showed a decrease in the levels of DOPAC (35.9%), 3-MT (24.7%) and 5-HIAA (9%). Hippocampus and striatum that received intraperitoneal injection of strictosidinic acid (10 mg/kg) showed 5-HT levels reduction of 63.4 e 28.7%, respectively. The biogenic amine levels alterations in the studied structures, associated with the analgesic and acetylcholinesterase inhibitor activities, suggest that P. myriantha and species from the Heteropsychotria subgenus constitute a font of bioactive compounds in the treatment of central nervous system disturbs.

Insulin-like growth factor-I (IGF-I) is the only known potentially modifiable risk factor for prostate cancer. Intake of dietary protein, especially from dairy products, might also be associated with risk and with circulating IGF-I, but it is not clear if amino acids play a role in these relationships. Moreover, investigations of circulating concentrations of metabolites might reveal novel risk factors for prostate cancer. This thesis investigates plasma concentrations of amino acids and other metabolites in relation to protein intake, IGF-I, and prostate cancer risk using data from the European Prospective Investigation into Cancer and Nutrition (EPIC). To characterise plasma metabolite profile in men consuming markedly different amounts and types of animal products (meat-eaters, fish-eaters, vegetarians and vegans), cross-sectional analyses of 392 men in the EPIC-Oxford sub-cohort were conducted. Of 21 amino acids, six varied significantly by diet group, and the metabolite profile of vegans was different from those of other diet groups owing to lower concentrations of several glycerophospholipids and sphingolipids. In a case-control study nested within EPIC, with a mean follow-up time of seven years, the relationship of plasma metabolites with risk of prostate cancer overall, by time to diagnosis, by tumour characteristics, and with risk of prostate cancer death, was investigated. Data from 1,077 matched sets suggested that seven metabolites, from various classes, were associated with risk of prostate cancer overall (p < 0.05). After correction for multiple testing, 12 glycerophospholipids were inversely associated with risk of advanced prostate cancer (the strongest OR_1SD = 0.54; 95%CI: 0.40-0.72). In multivariate analyses, including data from 1,593 matched sets, principal component analysis (PCA) and treelet transform (TT) were used to identify patterns in metabolite profile, five of which were associated with risk of more aggressive tumour sub-types (high grade, advanced and aggressive disease) and/or prostate cancer death. There was a ≈ 50% lower risk of advanced and high grade prostate cancer in men with metabolite profiles characterised by high glycerophospholipids and sphingolipids (for advanced OR_{TT, top vs bottom third} = 0:48; 95%CI: 0:31-0:74), with similar results for high grade and PCA). To investigate if associations between protein intake and circulating IGF-I may be mediated by plasma amino acid concentrations, cross-sectional analyses of amino acid concentrations with protein intake and IGF-I concentrations were carried out in 1,697 and 1,142 control participants, respectively, from the nested case-control study. Dairy protein intake was positively associated with concentrations of branched-chain amino acids and several other essential amino acids, while plant protein intake was strongly associated only with histidine. Serum IGF-I was positively associated with arginine and inversely with ornithine and certain amino acid ratios. In conclusion, men with different dietary habits with respect to the consumption of protein types have different amino acid and metabolite profiles, and metabolite concentrations may be associated with risk of more high-risk prostate cancer sub-types (high grade, advanced and aggressive disease) and prostate cancer death. Further large-scale studies are needed to determine if metabolites play a role in aetiology or are markers of sub-clinical prostate cancer.

The intention of this dissertation was an assessment of biogenic amines (BA) and polyamines (PA) by the usage of high-performance liquid chromatography (HPLC), in the concrete histamine (HIM), tyramine (TYM), 2-phenyl ethylamine (PEA), tryptamine (TRM), spermine (SPM), spermidine (SPD) and putrescine (PUT) in Velvet Bolete ( Suillus variegatus, (Sw.) Kuntze) and Bay Bolete ( Boletus badius, Fr.). The dissertation was focused on the influence of kitchen treatments by the method of pasteurization, freezing and subsequent storage of treated mushrooms on the content of biogenic amines and polyamines. Pasteurized samples were analysed immediately after adjustment on day 0 and then after three, six, nine and twelve months after storage in the dark place at 22 °C. The highest concentration in the pasteurized Velvet Bolete sample was observed in putrescine (1079 mg/kg). In the process of the experiment, this one and other biogenic amines levels decreased, except for tyramine, which showed a slight increase. The highest concentration in pasteurized Bay Bolete sample was also observed in putrescine (768 mg/kg) which, together with other biogenic amines levels, decreased. The frozen samples were stored at -16 °C and samples were taken every third, sixth, ninth and dozenth month. Changes in the concentration of biogenic amines and polyamines in the frozen sample of Velvet Bolete were identical with changes during pasteurization. Changes in the concentration of biogenic amines and polyamines in frozen sample of Bay Bolete were different from the pasteurized sample. Tryptamine, putrescine, spermidine, and spermine levels increased steadily, whereas the 2-phenylethylamine and tyramine levels decreased slightly. The monitoring results show that not even a unprofessional method of preserving wild mushrooms will lead to a substantial increase in the levels of biogenic amines and polyamines in the material of the wildly growing edible mushrooms.

The aim of this thesis was to determine the content of biogenic amines (BA)and polyamines (PA)in selected types of cheese and the influence of storage time on the content of biogenic amines.

O gênero Psychotria destaca-se na família RUBIACEAE pela produção de alcalóides bioativos e por sua taxonomia complexa, sendo muitas vezes relacionado aos gêneros Palicourea, Cephaelis, Calycodendron e Calycosia. A divisão de Psychotria nos subgêneros Psychotria, Tetramerae e Heteropsychotria foi proposta com o objetivo de auxiliar a classificação quimiotaxonômica do gênero. Estudos demonstram que o subgênero Psychotria (espécies pantropicais) produz alcalóides poliméricos, formados por duas ou mais unidades de triptamina; enquanto o subgênero Heteropsychotria parece estar envolvido com a produção de alcalóides indol monoterpênicos, de acordo com pesquisas realizadas com diferentes espécies coletadas no Sul do Brasil. O isolamento e purificação dos alcalóides estrictosamida, ácido estrictosidínico e miriantosina, a partir de Psychotria myriantha, corroboram com esta hipótese, permitindo a inclusão da espécie no subgênero Heteropsychotria. A literatura descreve várias atividades para extratos e alcalóides isolados de espécies de Psychotria, como antimicrobiana e analgésica, por exemplo. Neste trabalho, o extrato n-butanólico de alcalóides de P. myriantha, além de seus alcalóides isolados, apresentaram atividade inibidora da migração de leucócitos, sugerindo um efeito antiinflamatório, e capacidade de inibir a ação da enzima acetilcolinesterase. Extratos e alcalóides isolados da espécie foram avaliados quanto à atividade antioxidante em CCD, frente ao DPPH, apresentando resultado negativo. O extrato EBA e o alcalóide ácido estrictosidínico aumentaram o tempo de latência no teste da retirada da cauda frente ao estímulo térmico, indicando uma atividade analgésica do tipo opióide. A influência do ácido estrictosidínico, alcalóide isolado em maior quantidade em massa, sobre os níveis de DA, DOPAC, 3-MT, HVA, 5-HT e 5-HIAA em estruturas cerebrais de ratos foi verificada. Hipocampos de animais que receberam injeção intra-hipocampal bilateral de ácido estrictosidínico (20 μg/μL) apresentaram redução de 83,4 % nos níveis de serotonina, em comparação ao grupo controle, enquanto os córtices desses animais apresentaram redução nos níveis de DOPAC (35,9%), 3-MT (24,7%) e 5-HIAA (9%). Hipocampos e estriados de ratos tratados com injeção i.p. de ácido estrictosidínico (10 mg/kg) demonstraram diminuição de 63,4 e 28,7% nos níveis de 5-HT, respectivamente. As alterações nos níveis de aminas biogênicas nas estruturas avaliadas, além das atividades analgésica e inibidora da acetilcolinesterase, indicam que P. myriantha e espécies do subgênero Heteropsychotria constituem uma potencial fonte de substâncias bioativas no tratamento de distúrbios do sistema nervoso central. / Psychotria genus is an important in RUBIACEAE due to bioactive alkaloids production and complex taxonomy, being related to Palicourea, Cephaelis, Calycodendron and Calycosia genera. The division of Psychotria in Psychotria, Tetramerae and Heteropsychotria subgenera was proposed with the aim of aiding the genus chemotaxonomic classification. Studies demonstrate that Psychotria subgenus produce polyindoline alkaloids formed by two or more triptamine units; whereas Heteropsychotria subgenus seems to be involved with indole monoterpene alkaloids production, according to researches with different species collected in Southern Brazil. Isolation and purification of strictosamide, strictosidinic acid and miriantosine from Psychotria myriantha corroborated this hypothesis, allowing its inclusion into Heteropsychotria subgenus. The scientific literature describes several activities to Psychotria extracts and alkaloids, such as antimicrobial and analgesic, for example. In this work, P. myriantha n-butanolic alkaloid extract and isolated compounds inhibited the leukocyte migration, suggesting an antiinflammatory activity, and a weak ability to inhibit the action of acetylcholinesterase enzyme. Alkaloids and extracts from P. myriantha were evaluated as regards their antioxidant activity using DPPH, with no positive results. EBA and strictosidinic acid increased the latency in the tail flick model, indicating an opioid analgesic activity. The influence of strictosidinic acid on the levels of DA, DOPAC, 3-MT, HVA, 5-HT and 5-HIAA in brain structures of rats was verified. Hippocampus with intra-hippocampal injection of strictosidinic acid (20 μg/μL) displayed a decrease of 83.4% in serotonin levels, in comparison with control group; whereas the cortex showed a decrease in the levels of DOPAC (35.9%), 3-MT (24.7%) and 5-HIAA (9%). Hippocampus and striatum that received intraperitoneal injection of strictosidinic acid (10 mg/kg) showed 5-HT levels reduction of 63.4 e 28.7%, respectively. The biogenic amine levels alterations in the studied structures, associated with the analgesic and acetylcholinesterase inhibitor activities, suggest that P. myriantha and species from the Heteropsychotria subgenus constitute a font of bioactive compounds in the treatment of central nervous system disturbs.