The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA), a non-protein amino acid, first attracted attention in correlation to reports of high incidence of the unusual neurological disease amyotrophic lateral sclerosis/Parkinsonism-dementia (ALS/PDC) among the people of Guam in the South Pacific Ocean. Experimental studies have revealed that BMAA causes neuronal cell death. The neurotoxin is suggested to act via excitotoxicity through interaction with glutamatergic receptors. More importantly, BMAA is suggested to misincorporate in the synthesis of proteins, and contribute to protein misfolding and/or deleterious aggregation, which are hallmarks of several neurodegenerative disorders. A selective uptake of BMAA in the rat neonatal hippocampus can interfere with brain development, causing learning and memory impairments in adult rats. The aim of the present study was to investigate the effects of BMAA in human neuroblastoma SH-SY5Y cells. These cells were exposed to BMAA (10 μM, 50 μM, 100 μM or 500 μM) for 72 hours, and the expression of five selected proteins, including heat shock protein-27 (HSP-27), lysosomal associated membrane protein-1 (LAMP-1), CCAAT-enhancer-binding protein homologous protein (CHOP), Golgi associated plant pathogenesis related protein-2 (GLIPR-2), and glucose regulated protein-78 (GRP-78). They were carried out with immunohistochemistry (IHC). Results revealed an increased expression of all selected proteins, which indicates an uptake and shows the effects of BMAA in the cell cultures. Taken together, BMAA caused cellular stress, including endoplasmic reticulum (ER) stress that is correlated with HSP-27, LAMP-1, CHOP, GLIPR-2, and GRP-78. Further studies are needed in order to support the results. The experiments require being repeated using the same biomarkers as well as a combination of them with other biomarkers to elucidate the effects of BMAA.

Gunnera é um gênero de angiosperma pertencente à família Gunneraceae. O gênero possui aproximadamente 45 espécies, dentre essas G. perpensa L. tem sido utilizada na África, apresentando efeitos relacionados ao ciclo reprodutivo, provavelmente devido ao (Z)-venusol. Decocções dessa planta são utilizadas no tratamento de doenças infecciosas devido à atividade antibacteriana e à antifúngica. O gênero Gunnera é capaz de formar uma complexa associação com a cianobactéria Nostoc puctiforme L. ocorrendo a formação da neurotoxina -Nmetilamino- L-alanina (BMAA) a qual é associada ao aparecimento de um complexo de esclerose/Parkinson/demência. No Rio Grande do Sul, encontra-se a espécie G. manicata L., da qual não constam, na literatura científica, estudos fitoquímicos, farmacológicos e toxicológicos, apesar de ser apreciada ornamentalmente. Tendo em vista esses dados, o presente projeto avaliou o perfil tóxico-farmacológico e fitoquímico de G. manicata. Para os ensaios de toxicidade oral aguda e uterotrófico foi utilizado extrato aquoso de raízes e para as atividades antimicrobiana (disco difusão) e antioxidante (reação com 2,2 difenil-1-picril-hidrazil - DPPH•), bem como para o perfil fitoquímico, foram utilizados extratos metanólicos e aquosos de raízes e folhas. Não foram observados sinais de toxicidade oral aguda, nem atividade sob o sistema reprodutor de ratas. O potencial antimicrobiano resultou em atividade frente à Bacillus subtilis, Staphylococus aureus, Streptococcus pyogenes e Candida albicans. A capacidade antioxidante apresentou boa a moderada atividade. Na análise fitoquímica por cromatografia gasosa acoplada a detector de massa (CGEM) e cromatografia líquida associada à espectrometria de massas em tandem (CLEM/ EM) não foram detectados BMAA nem (Z)-venusol. Entretanto, foram identificados ácido gálico e hiperosídeo. Os extratos avaliados demonstraram alto teor de compostos fenólicos totais, apesar da baixa concentração de flavonóides totais. Sendo assim, os resultados obtidos neste trabalho geram perspectivas para novos estudos, tendo em vista à ausência de sinais de toxicidade, o potencial biológico apresentado, a falta de informações sobre G. manicata na literatura científica e a prospecção de espécies bioativas. / The genus Gunnera is a flowering plant belonging to the Gunneraceae family. The genus has about 45 species, among these G. Perpensa which has been used in Africa. It has effects related to the reproductive cycle, probably due to the (Z)- venusol. Decoctions of this plant are used in the treatment of infectious diseases due to antibacterial and antifungal activities. The genus Gunnera forms a complex association with the cyanobacterium Nostoc puctiforme L. resulting in the formation of the neurotoxin -N-methylamino-L-alanine (BMAA), which is associated with the development of a complex sclerosis / Parkinson / dementia. In Southern Brazil is found the specie Gunnera manicata L. on which scientific data about phytochemical, pharmacological and toxicological studies are lacking, despite of being assessed as ornamental plant. So, were investigated the toxico-pharmacological and phytochemical profile of G. manicata. In the oral acute toxicity and in the uterotrophic assays aqueous roots extracts were used. To antimicrobial and antioxidant (2,2- diphenyl-1-picryhydrazyl – DPPH - radical test) activities as well as phytochemical profile, methanol and aqueous extracts of roots and leaves were used. Was not observed neither sings of oral acute toxicity nor activity on the female reproductive system of rats. The antimicrobial potential showed activity against Bacillus subtilis, Staphylococus aureus, Streptococcus pyogenes and Candida albicans. Antioxidant capacity presented good to moderate activity. The phytochemical profile analysis was performed by gas chromatography coupled with mass spectrometer (GC-MS) and liquid chromatography coupled with tandem mass spectrometer (LC-MS/MS). BMAA and (Z)-venusol were not found and the samples presented gallic acid and hyperoside. The evaluated extracts showed a high content of total phenolic compounds despite of the low contends of total flavonoids. So, the results obtained in this work open prospects for new studies in view of the absence of signs of toxicity, the biological potential presented, the lack of information about G. manicata the scientific literature and the search for bioactive species.

β-N-methylamino-L-alanine (BMAA) is one of the more recently discovered bioactive compounds produced by cyanobacteria. BMAA is a non-protein amino acid reported present in human brain tissues of patients deceased from a neurodegenerative disease, such as Alzheimer´s disease or amyotrophic lateral sclerosis (ALS). This observation in combination with its neurotoxic effects in eukaryotes (in vivo and in vitro) and its potential to incorporate into (human) proteins, causing protein aggregation, suggests BMAA as a possible causative environmental agent for neurodegenerative diseases. Due to the ubiquitous nature of cyanobacteria with a wide occurrence in both aquatic and terrestrial environments, BMAA could be globally spread. Hence, investigations of a possible coupling between BMAA and neurodegeneration are urgently needed as well as to identify sources of BMAA in Nature. The aim of this thesis was to examine the potential occurrence of BMAA in bloom forming cyanobacteria of the Baltic Sea and its possible transfer to other organisms of this ecosystem. Of importance was also to reveal any likely routes for human BMAA exposure in the Baltic Sea region and to further investigate BMAA as a triggering agent for neurodegenerative diseases. Acknowledged difficulties of analysing BMAA in biological samples also inferred method development as part of the experimental studies. Investigating the role of BMAA in its producers was another purpose of the thesis, which may be crucial for future management of BMAA-producing cyanobacteria. By screening natural populations of the major filamentous bloom forming cyanobacteria of the Baltic Sea, we discovered the presence of BMAA throughout the entire summer season of two consecutive years, using a highly specific analytical method (liquid chromatography-tandem mass spectrometry; LC-MS/MS). BMAA was found to bioaccumulate in zooplankton and fish, as well as in mussels and oysters from the Swedish west coast. To improve the understanding of BMAA analyses in natural samples, the formation of carbamate adducts in the presence of bicarbonate was examined. Using two derivatization techniques in combination with LC-MS/MS, we could show that BMAA detection was not hindered by carbamate formation. Exogenously added BMAA inhibited nitrogen fixation in the model cyanobacterium Nostoc sp. PCC 7120, which was also hampered in growth and displayed signs of nitrogen starvation. Finally, BMAA was detected in cerebrospinal fluid in three of 25 Swedish test individuals, and represents the first confirmation of BMAA in the human central nervous system using LC-MS/MS as the primary analytical method. However, the association of BMAA to neurodegenerative diseases could not be verified as BMAA was present in both control individuals (two) and in one ALS-patient. Nevertheless, the finding of a known neurotoxic compound in the human central nervous system is alarming and potential consequences should be investigated. The discovery of the neurotoxic compound BMAA in Baltic Sea organisms, and in the central nervous system of humans potentially consuming fish from this ecosystem is concerning and warrants continued investigations of BMAA occurrence and human exposure. Further knowledge on the function and regulation of BMAA may help in developing strategies aiming to minimise human exposure. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

The foci of this dissertation are on advanced liquid chromatography (LC) separation and mass spectrometry (MS) techniques for the analysis of small bioactive molecules. In addition to discussing general aspects of such techniques the results from analyses of polyphenols (PPs), alkaloids and amino acids published in five appended studies are presented and discussed. High efficiency and well understood principles make LC the method of choice for separating analytes in many kinds of scientific investigations. Moreover, when LC is coupled to an MS instrument, analytes are separated in two stages: firstly they are separated and pre-concentrated in narrow bands using LC and then separated according to their mass-to-charge (m/z) ratios in the MS instrument. Some MS instruments can provide highly accurate molecular weight measurements and mass resolution allowing identification of unknown compounds based purely on MS data, thus making prior separation unnecessary. However, prior separation is essential for analyzing substances in most complex matrices – especially useful is the ultra-high performance LC (UHPLC). The advantages of using UHPLC rather than HPLC for the analysis of PPs in tea and wine were evaluated in one of the studies this thesis is based upon. The phenolic composition of red wine was also examined, using a novel LDI technique, following solid phase extraction (SPE). A class of small aromatic molecules (medicinally important alkaloids) also proved to be amenable to straightforward analysis, by thin layer chromatography (TLC) work-up followed by LDI-MS. Finally, a LC-MS method for monitoring neurotoxins (β-N-methyl-amino-L-alanine and 2,3-diaminobutyric acid) in complex biological matrices was developed and applied. Overall, the studies show that careful attention to the physicochemical properties of analytes can provide insights that can greatly facilitate the development of alternative methods to analyze them, e.g. by LDI. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: In press. Paper 5: Manuscript.</p>

Gunnera é um gênero de angiosperma pertencente à família Gunneraceae. O gênero possui aproximadamente 45 espécies, dentre essas G. perpensa L. tem sido utilizada na África, apresentando efeitos relacionados ao ciclo reprodutivo, provavelmente devido ao (Z)-venusol. Decocções dessa planta são utilizadas no tratamento de doenças infecciosas devido à atividade antibacteriana e à antifúngica. O gênero Gunnera é capaz de formar uma complexa associação com a cianobactéria Nostoc puctiforme L. ocorrendo a formação da neurotoxina -Nmetilamino- L-alanina (BMAA) a qual é associada ao aparecimento de um complexo de esclerose/Parkinson/demência. No Rio Grande do Sul, encontra-se a espécie G. manicata L., da qual não constam, na literatura científica, estudos fitoquímicos, farmacológicos e toxicológicos, apesar de ser apreciada ornamentalmente. Tendo em vista esses dados, o presente projeto avaliou o perfil tóxico-farmacológico e fitoquímico de G. manicata. Para os ensaios de toxicidade oral aguda e uterotrófico foi utilizado extrato aquoso de raízes e para as atividades antimicrobiana (disco difusão) e antioxidante (reação com 2,2 difenil-1-picril-hidrazil - DPPH•), bem como para o perfil fitoquímico, foram utilizados extratos metanólicos e aquosos de raízes e folhas. Não foram observados sinais de toxicidade oral aguda, nem atividade sob o sistema reprodutor de ratas. O potencial antimicrobiano resultou em atividade frente à Bacillus subtilis, Staphylococus aureus, Streptococcus pyogenes e Candida albicans. A capacidade antioxidante apresentou boa a moderada atividade. Na análise fitoquímica por cromatografia gasosa acoplada a detector de massa (CGEM) e cromatografia líquida associada à espectrometria de massas em tandem (CLEM/ EM) não foram detectados BMAA nem (Z)-venusol. Entretanto, foram identificados ácido gálico e hiperosídeo. Os extratos avaliados demonstraram alto teor de compostos fenólicos totais, apesar da baixa concentração de flavonóides totais. Sendo assim, os resultados obtidos neste trabalho geram perspectivas para novos estudos, tendo em vista à ausência de sinais de toxicidade, o potencial biológico apresentado, a falta de informações sobre G. manicata na literatura científica e a prospecção de espécies bioativas. / The genus Gunnera is a flowering plant belonging to the Gunneraceae family. The genus has about 45 species, among these G. Perpensa which has been used in Africa. It has effects related to the reproductive cycle, probably due to the (Z)- venusol. Decoctions of this plant are used in the treatment of infectious diseases due to antibacterial and antifungal activities. The genus Gunnera forms a complex association with the cyanobacterium Nostoc puctiforme L. resulting in the formation of the neurotoxin -N-methylamino-L-alanine (BMAA), which is associated with the development of a complex sclerosis / Parkinson / dementia. In Southern Brazil is found the specie Gunnera manicata L. on which scientific data about phytochemical, pharmacological and toxicological studies are lacking, despite of being assessed as ornamental plant. So, were investigated the toxico-pharmacological and phytochemical profile of G. manicata. In the oral acute toxicity and in the uterotrophic assays aqueous roots extracts were used. To antimicrobial and antioxidant (2,2- diphenyl-1-picryhydrazyl – DPPH - radical test) activities as well as phytochemical profile, methanol and aqueous extracts of roots and leaves were used. Was not observed neither sings of oral acute toxicity nor activity on the female reproductive system of rats. The antimicrobial potential showed activity against Bacillus subtilis, Staphylococus aureus, Streptococcus pyogenes and Candida albicans. Antioxidant capacity presented good to moderate activity. The phytochemical profile analysis was performed by gas chromatography coupled with mass spectrometer (GC-MS) and liquid chromatography coupled with tandem mass spectrometer (LC-MS/MS). BMAA and (Z)-venusol were not found and the samples presented gallic acid and hyperoside. The evaluated extracts showed a high content of total phenolic compounds despite of the low contends of total flavonoids. So, the results obtained in this work open prospects for new studies in view of the absence of signs of toxicity, the biological potential presented, the lack of information about G. manicata the scientific literature and the search for bioactive species.

Gunnera é um gênero de angiosperma pertencente à família Gunneraceae. O gênero possui aproximadamente 45 espécies, dentre essas G. perpensa L. tem sido utilizada na África, apresentando efeitos relacionados ao ciclo reprodutivo, provavelmente devido ao (Z)-venusol. Decocções dessa planta são utilizadas no tratamento de doenças infecciosas devido à atividade antibacteriana e à antifúngica. O gênero Gunnera é capaz de formar uma complexa associação com a cianobactéria Nostoc puctiforme L. ocorrendo a formação da neurotoxina -Nmetilamino- L-alanina (BMAA) a qual é associada ao aparecimento de um complexo de esclerose/Parkinson/demência. No Rio Grande do Sul, encontra-se a espécie G. manicata L., da qual não constam, na literatura científica, estudos fitoquímicos, farmacológicos e toxicológicos, apesar de ser apreciada ornamentalmente. Tendo em vista esses dados, o presente projeto avaliou o perfil tóxico-farmacológico e fitoquímico de G. manicata. Para os ensaios de toxicidade oral aguda e uterotrófico foi utilizado extrato aquoso de raízes e para as atividades antimicrobiana (disco difusão) e antioxidante (reação com 2,2 difenil-1-picril-hidrazil - DPPH•), bem como para o perfil fitoquímico, foram utilizados extratos metanólicos e aquosos de raízes e folhas. Não foram observados sinais de toxicidade oral aguda, nem atividade sob o sistema reprodutor de ratas. O potencial antimicrobiano resultou em atividade frente à Bacillus subtilis, Staphylococus aureus, Streptococcus pyogenes e Candida albicans. A capacidade antioxidante apresentou boa a moderada atividade. Na análise fitoquímica por cromatografia gasosa acoplada a detector de massa (CGEM) e cromatografia líquida associada à espectrometria de massas em tandem (CLEM/ EM) não foram detectados BMAA nem (Z)-venusol. Entretanto, foram identificados ácido gálico e hiperosídeo. Os extratos avaliados demonstraram alto teor de compostos fenólicos totais, apesar da baixa concentração de flavonóides totais. Sendo assim, os resultados obtidos neste trabalho geram perspectivas para novos estudos, tendo em vista à ausência de sinais de toxicidade, o potencial biológico apresentado, a falta de informações sobre G. manicata na literatura científica e a prospecção de espécies bioativas. / The genus Gunnera is a flowering plant belonging to the Gunneraceae family. The genus has about 45 species, among these G. Perpensa which has been used in Africa. It has effects related to the reproductive cycle, probably due to the (Z)- venusol. Decoctions of this plant are used in the treatment of infectious diseases due to antibacterial and antifungal activities. The genus Gunnera forms a complex association with the cyanobacterium Nostoc puctiforme L. resulting in the formation of the neurotoxin -N-methylamino-L-alanine (BMAA), which is associated with the development of a complex sclerosis / Parkinson / dementia. In Southern Brazil is found the specie Gunnera manicata L. on which scientific data about phytochemical, pharmacological and toxicological studies are lacking, despite of being assessed as ornamental plant. So, were investigated the toxico-pharmacological and phytochemical profile of G. manicata. In the oral acute toxicity and in the uterotrophic assays aqueous roots extracts were used. To antimicrobial and antioxidant (2,2- diphenyl-1-picryhydrazyl – DPPH - radical test) activities as well as phytochemical profile, methanol and aqueous extracts of roots and leaves were used. Was not observed neither sings of oral acute toxicity nor activity on the female reproductive system of rats. The antimicrobial potential showed activity against Bacillus subtilis, Staphylococus aureus, Streptococcus pyogenes and Candida albicans. Antioxidant capacity presented good to moderate activity. The phytochemical profile analysis was performed by gas chromatography coupled with mass spectrometer (GC-MS) and liquid chromatography coupled with tandem mass spectrometer (LC-MS/MS). BMAA and (Z)-venusol were not found and the samples presented gallic acid and hyperoside. The evaluated extracts showed a high content of total phenolic compounds despite of the low contends of total flavonoids. So, the results obtained in this work open prospects for new studies in view of the absence of signs of toxicity, the biological potential presented, the lack of information about G. manicata the scientific literature and the search for bioactive species.

No description available.

Many cyanobacteria are reported to produce the nonprotein amino acid β-N-methylamino-L-alanine (BMAA). Cyanobacteria are extensively distributed in terrestrial and aquatic environments and recently BMAA was detected in temperate aquatic ecosystems, e.g. the Baltic Sea. Little is known about developmental effects of the mixed glutamate receptor agonist BMAA. Brain development requires an optimal level of glutamate receptor activity as the glutamatergic system modulates many vital neurodevelopmental processes. The aim of this thesis was to investigate the developmental neurotoxicity of BMAA, and its interaction with the pigment melanin. Autoradiography was utilized to determine the tissue distribution of 3H-labelled BMAA in experimental animals. Behavioral studies and histological techniques were used to study short and long-term changes in the brain following neonatal exposure to BMAA. Long-term changes in protein expression in the brain was also investigated using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). A notable targeting of 3H-BMAA to discrete brain regions e.g. hippocampus and striatum in mouse fetuses and neonates was determined by autoradiography. BMAA treatment of neonatal rats on postnatal days 9–10 induced acute but transient ataxia and hyperactivity. Postnatal exposure to BMAA also gave rise to reduced spatial learning and memory abilities in adulthood. Neonatal rat pups treated with BMAA at 600 mg/kg showed early neuronal cell death in the hippocampus, retrosplenial and cingulate cortices. In adulthood the CA1 region of the hippocampus displayed neuronal loss and astrogliosis. Lower doses of BMAA (50 and 200 mg/kg) caused impairments in learning and memory function without any acute or long-term morphological changes in the brain. The MALDI IMS studies, however, revealed changes in protein expression in the hippocampus and striatum suggesting more subtle effects on neurodevelopmental processes. The studies also showed that BMAA was bound and incorporated in melanin and neuromelanin, suggesting that pigmented tissues such as in the substantia nigra and eye may be sequestering BMAA. In conclusion, the findings in this thesis show that BMAA is a developmental neurotoxin in rodents. The risks posed by BMAA as a potential human neurotoxin merits further consideration, particularly if the proposed biomagnifications in the food chain are confirmed.

Beta-N-methylamino-L-alanine (BMAA) is a non-protein amino acid produced by many cyanobacteria, and thought to induce neurotoxic effects through excitotoxicity, contributing to neurodegenerative diseases such as Amyotrophic Lateral Sclerosis/Parkinsonism-dementia complex (ALS-PDC) and Alzheimer’s. The ubiquitous nature of cyanobacteria, and evidence of biomagnification through our food web, creates a dire need for the development of an analytical platform that will provide accurate identification and quantification of BMAA amounts in our ecosystem and potential food supply. The present study evaluated the ability of a MALDI-ToF-MS method to detect and quantify BMAA in a variety of biological matrices. Through validation procedures, it was demonstrated that this MALDI-ToF-MS method provided comparable data to currently accepted analytical methods, specifically LC-MS/MS. Further, the development of said method reduced sample preparation and data acquisition time (1-2 seconds per sample), while providing high throughput analysis and eliminating the need for derivatization, chromatographic separation, and modification of amino acids.

A selective neurotoxin takes many forms: as an antibody to a neurotrophin, as an alkylator, as an excitotoxin, as a blocker of requisite neuronal excitation during ontogenetic development, as a generator of oxidative stress, as an inhibitor of vital intraneuronal processes, and as an agent adversely affecting a host of multiple sites in neurons. Neurotoxins have been invaluable for elucidating cellular mechanisms attending or preventing neuronal necrosis and apoptosis, and for modeling and thereby discerning mechanisms invoked in neurological and psychiatric disorders. Neuroprotectants, endogenous and exogenous, are being explored as potentially useful agents to ward off diseases. Finally, hypothesized as posing a risk to humans as environmental constituents, neurotoxins are now being remodeled as adjuncts for therapeutic intervention in a variety of human medical disorders.