Os inibidores da enzima acetilcolinesterase constituem o principal tratamento da doença de Alzheimer e fontes de substâncias naturais com potencial anticolinesterásico vêm sendo amplamente estudadas. Dentre os frutos com benefícios para a saúde, a romã é evidenciada como excelente fonte de compostos antioxidantes, sendo que maior parte dos compostos se concentram em sua casca. Com base nisso, o objetivo desta pesquisa foi buscar novas substâncias naturais com potencial anticolinesterásico, através da avaliação de extratos de casca de romã. Quatro extratos com diferentes concentrações de etanol foram analisados quanto à atividade antioxidante, quantidade de compostos fenólicos, taninos e atividade anticolinesterásica. Do presente estudo foi constatado que a casca da romã apresentou elevada capacidade antioxidante, independente da concentração do solvente de extração empregado. O extrato formulado com 80% de etanol se destacou perante os demais pelo seu poder de inibição da acetilcolinesterase. Houve correlação negativa entre a atividade anticolinesterásica e a atividade antioxidante dos extratos. A atomização do extrato não acarretou mudanças em sua atividade anticolinesterásica e nem na sua capacidade antioxidante. Da mesma forma, a adição das micropartículas a um suco elaborado a partir de um preparado em pó não modificou suas características sensoriais. Diante do exposto, a elaboração de micropartículas de extrato de casca de romã constitui alternativa viável para a incorporação em diversos produtos, com a finalidade de prevenir ou reduzir risco da doença de Alzheimer. / The acetylcholinesterase inhibitors are the primary treatment of Alzheimer\'s disease and sources of natural substances with potential anticholinesterase have been widely studied. Among the fruits with health benefits, the pomegranate is evidenced as an excellent source of antioxidant compounds, and most compounds are concentrated in its peel. Based on this, the objective of this research was to find new substances with potential anticholinesterase, through the evaluation of pomegranate peel extracts. Four extracts with different concentrations of ethanol were analyzed for their antioxidant activity, amount of phenolic compounds, tannins and anticholinesterase activity. From this study it was found that pomegranate peel showed high antioxidant capacity, independent of the concentration of the solvent extraction employed. The extract formulated with 80% ethanol in relation to other stood out by his power of acetylcholinesterase inhibition. There was a negative correlation between acetylcholinesterase activity and antioxidant activity of the extracts. Microencapsulation of extract did not cause changes in their anticholinesterase activity and antioxidant capacity. The same way, the addition of microcapsules to a powder preparation for refreshment not changed their sensory characteristics. Given the above, the preparation of microcapsules of pomegranate peel extract is a viable alternative for incorporation into various products, in order to prevent or reduce risk of Alzheimer\'s disease.

Aldeídos de colesterol (Secosterol A e Secosterol B) têm sido detectados em amostras de cérebro humano e investigados em modelos de doenças neurodegenerativas como possíveis marcadores e intermediários do processo patológico. Estes oxisteróis constituem uma classe de eletrófilos derivados de lipídeos que podem modificar e induzir agregação de proteínas. A esclerose lateral amiotrófica (ELA) é um distúrbio neurodegenerativo associado ao acúmulo de agregados imunorreativos de superóxido dismutase (Cu, Zn-SOD, SOD1). O objetivo deste trabalho foi avaliar a presença de aldeídos de colesterol em ratos modelo ELA e sua capacidade de induzir a formação de agregados de SOD1 in vitro. Aldeídos de colesterol foram analisados no plasma, medula espinhal e córtex motor de ratos ELA. Uma quantidade elevada de Secosterol B foi detectada no córtex motor desses ratos em comparação com animais controle. Adicionalmente, os experimentos in vitro mostraram que Secosterol B e Secosterol A induziram a agregação da SOD1 em uma forma amiloidogênica que se liga à tioflavina T. Esta agregação não foi observada com o colesterol e os seus hidroperóxidos. Usando aldeídos de colesterol marcados com grupo alquinil e um ensaio de click chemistry, foi observado que os agregados de SOD1 estão ligados covalentemente aos aldeídos. A modificação covalente da proteína foi confirmada por análise de MALDI-TOF, que mostrou a adição de até cinco moléculas de aldeídos de colesterol à proteína por base de Schiff. Curiosamente, a análise comparativa com outros eletrófilos derivados de lipídeos (e.g. HHE e HNE) demonstrou que a agregação de SOD1 aumentou proporcionalmente à hidrofobicidade dos aldeídos, observando-se a maioragregação com aldeídos de colesterol. Os sítios de modificação da SOD1 foram caracterizados por nanoLC-MS/MS após digestão da proteína com tripsina, onde foram identificadas lisinas como o principal aminoácido modificado. Em geral, nossos dados mostram que a oxidação do colesterol que leva à produção de aldeídos de colesterol é aumentada no cérebro de ratos ELA e que os aldeídos altamente hidrofóbicos derivados de colesterol podem promover eficientemente modificação e agregação de SOD1. / Secosterol aldehydes (Secosterol B and Secosterol A) have been detected in human brain samples and investigated in models of neurodegenerative diseases as possible markers and intermediates of the pathological process. These oxysterols constitute a class of lipid-derived electrophiles that can modify and induce aggregation of proteins. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with the accumulation of immunoreactive aggregates of superoxide dismutase (Cu, Zn-SOD, SOD1). The objective of this work is to evaluate the presence of secosterol aldehydes in ALS rats and their ability to induce formation of SOD1 aggregates in vitro. Secosterol aldehydes were analyzed in plasma, spinal cord and motor cortex of ALS rats. A higher amount of Secosterol B was detected in the motor cortex of these rats compared to control animals. In addition, in vitro experiments have shown that Secosterol B and Secosterol A induce aggregation of SOD1 into an amyloidogenic form that binds to thioflavin T. This aggregation was not apparent in incubations with cholesterol and its hydroperoxides. Using alkynyl-labeled secosterol aldehydes and a click chemistry assay, it was found that the SOD1 aggregates are covalently linked to the aldehydes. Covalent modification of the protein was confirmed by MALDI-TOF analysis, which showed the addition of up to five molecules of secosterol aldehydes to the protein by Schiff base formation. Interestingly, the comparative analysis with other lipid-derived electrophiles (e.g. HHE and HNE) demonstrated that the aggregation of SOD1 increased according to the hydrophobicity of the aldehydes. Compared to the other electrophiles, a higher SOD1 aggregation was observed with secosterol aldehydes. SOD1 modification sites were characterized by nanoLC-MS/MS afterprotein digestion with trypsin, revealing lysine as the major amino acid modified in these experiments. Collectively, our data show that cholesterol oxidation leads to the production of secosterol aldehydes, which are increased in the brain of ALS rats, and that these highly hydrophobic aldehydes can efficiently promote the modification and aggregation of SOD1.

Introdução: A doença por grãos argirofílicos (DGA) é uma tauopatia esporádica distinta, bastante frequente, com uma prevalência atingindo 31,3% em centenários, porém pouco reconhecida A manifestação clínica mais comum da DGA é de um comprometimento cognitivo de lenta evolução associado a uma alta frequência de sintomas psiquiátricos. O diagnóstico de DGA é possível somente através da análise do encéfalo post-mortem com os achados das três principais alterações patológicas: grãos argirofílicos, corpúsculos em embrião e pré-emaranhados neuronais. O presente estudo investigou as características demográficas, clínicas e neuropatológicas dos indivíduos com DGA e possíveis associações clínico-patológicas. Métodos: Foram estudados 983 casos (acima de 50 anos de idade) provenientes da amostra do Banco de Encéfalos do Grupo de Estudos em Envelhecimento Cerebral. A avaliação clínica e funcional foi realizada através de uma ampla entrevista semiestruturada respondida por um informante com contato próximo com o paciente. Os participantes foram estratificados conforme a presença de comprometimento cognitivo (de acordo com Escala de Avaliação Clinica da Demência) e, posteriormente, pela presença de DGA em quatro grupos: DGA com e sem comprometimento cognitivo e não-DGA com e sem comprometimento cognitivo. Análise descritiva foi realizada para dados socioeconômicos, genótipo de APOE e variáveis clínico-funcionais, neuropsiquiátricas e neuropatológicas na amostra DGA e em cada grupo. Foi utilizado um modelo de regressão logística multivariada para investigar as associações entre perfil cognitivo e sintomas neuropsiquiátricos com DGA. Resultados: DGA foi identificada em 150 indivíduos (15,1%). Idade avançada e baixo nível socioeconômico foram associados com DGA independente da presença de comprometimento cognitivo. A presença de DGA foi associada a uma redução de 60% na probabilidade de um escore >= 3.8 no Questionário do informante sobre o Declínio Cognitivo do Idoso (OR=0,40; IC de 95% 0,22-0,74; p=0,004). Adicionalmente, o subitem apetite do inventário neuropsiquiátrico foi associado à DGA em indivíduos cognitivamente normais (OR=1,85; IC de 95% 1,09-3,12; p=0,02). Conclusão: A DGA pode preservar a cognição em indivíduos com patologias neurodegenerativas associadas em particular nos casos com patologia tipo Alzheimer concomitante. A investigação dos mecanismos subjacentes a esse efeito pode auxiliar no desenvolvimento de novos tratamentos para a Doença de Alzheimer / Background: Argyrophilic grain disease (AGD) is an underrecognized, distinct, highly frequent sporadic tauopathy, with prevalence reaching 31.3% in centenarians. The most common presentation of AGD is a slowly progressive amnestic mild cognitive impairment, accompanied by high frequency of neuropsychiatric symptoms. AGD can only be diagnosed postmortem by the finding of its three main pathologic features: argyrophilic grains, oligodendrocytic coiled bodies and neuronal pretangles. The present study investigated demographic, clinical, and neuropathological profiles and analyzed clinicopathological associations. Methods: We studied 983 participants (over 50 years of age) from the Brain Bank of the Brazilian Aging Brain study group sample. Clinical and functional evaluation included demographics and a semi-structured interview covering various cognitive domains conducted with a knowledgeable informant. Participants were stratified by cognitive status (based on Clinical Dementia Rating scale), followed by the presence of AGD in four groups: AGD with and without cognitive impairmet, and non-AGD with and without cognitive impairment. Descriptive statistics were used for sociodemographic data, APOE genotypes, and the clinical, cognitive, neuropsychiatric, functional, and neuropathological variables in AGD samples and in each group. We used multivariate logistic regression models to investigate the association between the cognitive status and neuropsychiatric symptoms with AGD. Results: AGD was identified in 150 participants (15.1%). Older age and lower socioeconomic status were associated with AGD independent of cognitive status. Multivariate analyses revealed that AGD was associated with a 60% reduction in the odds of having an IQCODE >= 3.8 (OR = 0.40, 95% CI 0.22-0.74, p = 0.004) and that the NPI sub-item \"appetite and eating abnormalities\" was associated with AGD in controls (OR = 1.85, 95% CI 1.09-3.12, p = 0.02). Conclusion: AGD might preserve cognition in individuals with coexistent neurodegenerative pathologies, in particular those of the Alzheimer-type. Investigating whether the mechanisms underlying this effect could provide novel therapeutic approaches to the treatment of Alzheimer´s disease

Aldeídos de colesterol (Secosterol A e Secosterol B) têm sido detectados em amostras de cérebro humano e investigados em modelos de doenças neurodegenerativas como possíveis marcadores e intermediários do processo patológico. Estes oxisteróis constituem uma classe de eletrófilos derivados de lipídeos que podem modificar e induzir agregação de proteínas. A esclerose lateral amiotrófica (ELA) é um distúrbio neurodegenerativo associado ao acúmulo de agregados imunorreativos de superóxido dismutase (Cu, Zn-SOD, SOD1). O objetivo deste trabalho foi avaliar a presença de aldeídos de colesterol em ratos modelo ELA e sua capacidade de induzir a formação de agregados de SOD1 in vitro. Aldeídos de colesterol foram analisados no plasma, medula espinhal e córtex motor de ratos ELA. Uma quantidade elevada de Secosterol B foi detectada no córtex motor desses ratos em comparação com animais controle. Adicionalmente, os experimentos in vitro mostraram que Secosterol B e Secosterol A induziram a agregação da SOD1 em uma forma amiloidogênica que se liga à tioflavina T. Esta agregação não foi observada com o colesterol e os seus hidroperóxidos. Usando aldeídos de colesterol marcados com grupo alquinil e um ensaio de click chemistry, foi observado que os agregados de SOD1 estão ligados covalentemente aos aldeídos. A modificação covalente da proteína foi confirmada por análise de MALDI-TOF, que mostrou a adição de até cinco moléculas de aldeídos de colesterol à proteína por base de Schiff. Curiosamente, a análise comparativa com outros eletrófilos derivados de lipídeos (e.g. HHE e HNE) demonstrou que a agregação de SOD1 aumentou proporcionalmente à hidrofobicidade dos aldeídos, observando-se a maioragregação com aldeídos de colesterol. Os sítios de modificação da SOD1 foram caracterizados por nanoLC-MS/MS após digestão da proteína com tripsina, onde foram identificadas lisinas como o principal aminoácido modificado. Em geral, nossos dados mostram que a oxidação do colesterol que leva à produção de aldeídos de colesterol é aumentada no cérebro de ratos ELA e que os aldeídos altamente hidrofóbicos derivados de colesterol podem promover eficientemente modificação e agregação de SOD1. / Secosterol aldehydes (Secosterol B and Secosterol A) have been detected in human brain samples and investigated in models of neurodegenerative diseases as possible markers and intermediates of the pathological process. These oxysterols constitute a class of lipid-derived electrophiles that can modify and induce aggregation of proteins. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with the accumulation of immunoreactive aggregates of superoxide dismutase (Cu, Zn-SOD, SOD1). The objective of this work is to evaluate the presence of secosterol aldehydes in ALS rats and their ability to induce formation of SOD1 aggregates in vitro. Secosterol aldehydes were analyzed in plasma, spinal cord and motor cortex of ALS rats. A higher amount of Secosterol B was detected in the motor cortex of these rats compared to control animals. In addition, in vitro experiments have shown that Secosterol B and Secosterol A induce aggregation of SOD1 into an amyloidogenic form that binds to thioflavin T. This aggregation was not apparent in incubations with cholesterol and its hydroperoxides. Using alkynyl-labeled secosterol aldehydes and a click chemistry assay, it was found that the SOD1 aggregates are covalently linked to the aldehydes. Covalent modification of the protein was confirmed by MALDI-TOF analysis, which showed the addition of up to five molecules of secosterol aldehydes to the protein by Schiff base formation. Interestingly, the comparative analysis with other lipid-derived electrophiles (e.g. HHE and HNE) demonstrated that the aggregation of SOD1 increased according to the hydrophobicity of the aldehydes. Compared to the other electrophiles, a higher SOD1 aggregation was observed with secosterol aldehydes. SOD1 modification sites were characterized by nanoLC-MS/MS afterprotein digestion with trypsin, revealing lysine as the major amino acid modified in these experiments. Collectively, our data show that cholesterol oxidation leads to the production of secosterol aldehydes, which are increased in the brain of ALS rats, and that these highly hydrophobic aldehydes can efficiently promote the modification and aggregation of SOD1.

Hepcidin is a well-known iron-regulatory hormone that plays a key role in maintaining peripheral iron homeostasis. The presence and wide-spread distribution of hepcidin in the brain suggests that this peptide may also be an important player in brain iron homeostasis. In this study, we tested the hypothesis that hepcidin exerts an important role in the regulation of brain iron content, which might benefit iron-associated NDs such as PD. We also examined the hypothesis that hepcidin could control iron transport processes via regulating iron transport proteins in the brain cells, thus maintaining brain iron homeostasis. / In conclusion, the results of the present study implied that hepcidin plays an important role in maintaining brain iron homeostasis. Hepcidin is beneficial for PD and this effect is related to its iron-regulatory effect via inhibiting iron accumulation in the substantia nigra. Hepcidin effectively controls iron uptake and release through regulating iron transport proteins expressions in the brain, which would contribute to brain iron homeostasis. Therefore, manipulation of hepcidin level in the brain has a potential to be developed into a novel preventive approach for the iron-associated NDs such as PD. / In the second part, we investigated the effect of hepcidin on the processes of iron uptake and release in the cultured brain cells including neurons, astrocytes and brain vascular endothelial cells (BVECs). The expressions of iron uptake proteins such as transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) as well as the iron exporter ferroportin 1 (Fpn1) were also observed. We found that hepcidin reduced both iron uptake and release via decreasing iron transport proteins expressions in these brain cells, which would contribute to its iron regulatory effect. Finally, we further explored the mechanisms underlying the regulatory effect of hepcidin on the iron transporters in the last part, and found that the action of hepcidin in reducing TfR1 expression is a direct and cAMP-PKA (Cyclic Adenosine 3', 5'-monophosphate/ Protein Kinase-A) pathway-dependent event. / Iron is a transition trace metal essential for mammalian cellular and tissue viability. It also plays important roles in the central nervous system (CNS), including embryonic brain development, myelination, and neurotransmitters synthesis. However, abnormal iron accumulation has been demonstrated in a number of neurodegenerative diseases (NDs) such as Parkinson's (PD), Alzheimer's (AD) and Huntington's diseases (HD). Currently very little is known about the mechanisms involved in brain iron homeostasis and therefore it is not known why and how iron is abnormally increased in the brain. However, given the essential role that excess iron plays in the pathological processes in the NDs, to suppress the accumulated iron is expected to be an effective strategy to prevent and treat these NDs. / To investigate whether hepcidin could benefit iron-associated NDs including PD and whether this beneficial role is related to its iron-regulatory function in the brain, in the first part of study, we investigated the effects of hepcidin on the 6-hydroxydopamine (6-OHDA) in vitro and in vivo PD models. We found that in primary cultured mesencephalic (MES) neurons, hepcidin overexpression via adenovirus-hepcidin (Ad-hepcidin) infection prevented 6-OHDA-induced increase in cellular iron content and protected the MES neurons. In the 6-OHDA model of PD in vivo, overexpression of hepcidin in the substantia nigra via Ad-hepcidin intranigral injection significantly prevented iron accumulation and dopaminergic neurons loss in the pars compacta of substantia nigra (SNc). These effects were accompanied by a marked improvement in motor performance of the PD animals. These findings indicate that hepcidin could benefit iron-associated NDs such as PD and via its iron-regulatory role in the brain. / Du, Fang. / Adviser: Ya Ke. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 152-173). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

De plus en plus de preuves montrent que le transport axonal rapide (FAT) joue un rôle crucial au cours des maladies neurodégénératives (NDs). La maladie de Huntington est une maladie neurodégénérative causée par une expansion anormale de polyglutamines dans la partie Nterminale de la protéine huntingtine (HTT) : une grande protéine d’échafaudage impliquée dans la régulation du transport. La présence de HTT mutante comme l’absence de la HTT induisent des défauts de transport chez les mammifères. Chez la Drosophile, la HTT mutante reproduit le phénotype observée chez les mammifères, cependant la fonction conservée de la HTT chez la Drosophile melanogaster (DmHTT) n’est pas encore clairement établie. Ici nous mettons en évidence que DmHTT s’associe aux vésicules, aux microtubules et intéragit avec la proteine dynéine. Dans les neurones corticaux de rat, DmHTT remplace partiellement la HTT de mammifère dans le transport axonal rapide, et les drosophiles invalidées pour la HTT montrent des défauts de transport axonal in vivo. Ces résultats suggèrent que la fonction de la HTT est conservée dans le modèle Drosophile.Le FAT est un processus qui requiert un apport constant d’énergie. Les mitochondries sont les principales sources de production d’ATP de la cellule. Cependant nous avons démontré que le FAT ne dépend non pas de cette source d’énergie là, contrairement à ce que l’on pensait, mais de l’ATP glycolytique produit par les vésicules. La dérégulation de GAPDH ou de PK, les deux enzymes glycolytiques productrices d’ATP, ralentit le transport vésiculaire. Néanmoins, l’invalidation de GAPDH n’affecte pas le transport mitochondrial. En outre, toutes les enzymes glycolytiques sont associées à des vésicules dynamiques et sont capables de produire leur propre ATP. Enfin nous montrons que l’ATP produit est suffisant pour assurer leur propre transport, prouvant l’autonomie énergétique des vésicules pour le transport. / Growing evidence support the idea that impairments in Fast Axonal Transport (FAT) play a crucial role in Neurodegenerative Diseases (NDs). Huntington’s Disease is neurodegenerative disorder caused by an abnormal polyglutamine expansion in the N-Terminal part of huntingtin (HTT), a large scaffold protein implicated in transport regulation. Both the presence of the mutated HTT as the loss of HTT leads to transport defects in mammals. In the fruit fly overexpression of the mutant HTT recapitulates the phenotype observed in mammals. However, it is still unclear whether HTT’s function is conserved in D. melanogaster. Here, we show that D. melanogaster HTT (DmHTT) associates with vesicles, microtubules, and interacts with dynein. In rat cortical neurons, DmHTT partially replaces mammalian HTT in fast axonal transport, and DmHTT KO flies show axonal transport defects in vivo. These results suggest that HTT function in transport is conserved in D. melanogaster.FAT is a process that requires a constant supply of energy. Mitochondria are the main producers of ATP in the cell. However, we have demonstrated that FAT does not depend on this source of energy, as previously thought, but it depends on glycolytic ATP produced on vesicles. Perturbing GAPDH or PK, the two ATP generating glycolytic enzymes, slows down vesicular transport. However, knocking down GAPDH does not affect mitochondrial transport. Furthermore, all of the glycolytic enzymes are associated with dynamic vesicles, and are capable of producing their own ATP. Finally, we show that this ATP production is sufficient to sustain their own transport, demonstrating the energetical autonomy of vesicles for transport.

Lam Wun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 140-153). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ACKNOWLDGEMENT --- p.iii / TABLE OF CONTENT --- p.iv / ABBREVIATIONS --- p.x / LISTS OF TABLES --- p.xi / LISTS OF FIGURES --- p.xii / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- Neurodegeneration and triplet repeat diseases --- p.1 / Chapter 1.2 --- Polyglutamine diseases --- p.2 / Chapter 1.3 --- Polyglutamine nuclear inclusions --- p.4 / Chapter 1.3.1 --- Kinetics of polyglutamine nuclear inclusion formation --- p.4 / Chapter 1.3.2 --- Roles of protein inclusions in neurodegeneration --- p.7 / Chapter 1.4 --- Polyglutamine pathogenic pathways --- p.8 / Chapter 1.4.1 --- Protein depletion theory --- p.9 / Chapter 1.4.2 --- Induction of apoptotic pathways --- p.13 / Chapter 1.5 --- Previous study on NI proteins --- p.14 / Chapter 1.6 --- Drosophila model for studying polyglutamine diseases --- p.15 / Chapter 1.6.1 --- Drosophila model for studying human diseases --- p.15 / Chapter 1.6.2 --- GAL4/UAS gene expression system --- p.15 / Chapter 1.6.3 --- Drosophila polyglutamine models --- p.17 / Chapter 1.7 --- Objectives of the study --- p.21 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Drosophila genetics --- p.22 / Chapter 2.1.1 --- Drosophila culture --- p.22 / Chapter 2.1.2 --- GAL4/UAS gene expression system --- p.22 / Chapter 2.1.3 --- Eye phenotypic analysis --- p.25 / Chapter 2.1.4 --- Polyglutamine fly models --- p.25 / Chapter 2.1.5 --- Generation and characterization of GFP-polyglutamine transgenic fly models --- p.25 / Chapter 2.2 --- Proteomic identification of nuclear inclusion proteins --- p.26 / Chapter 2.2.1 --- Proteomic identification of NI proteins by SDS-insolubility of NIs --- p.26 / Chapter 2.2.2 --- Proteomic identification of NI proteins by FA-solubility of NIs --- p.27 / Chapter 2.2.2.1 --- Approach overview --- p.27 / Chapter 2.2.2.2 --- Sample preparation for two-dimensional gel electrophoresis --- p.27 / Chapter 2.2.2.3 --- Two-dimensional gel electrophoresis --- p.29 / Chapter 2.2.2.4 --- Polyacrylamide gel staining --- p.31 / Chapter 2.2.2.5 --- Computer analysis of 2D patterns --- p.31 / Chapter 2.2.2.6 --- In-gel trypsin digestion --- p.32 / Chapter 2.2.2.7 --- Mass spectrometric analysis --- p.33 / Chapter 2.2.3 --- Detection of NIs by flow cytometry --- p.34 / Chapter 2.3 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.34 / Chapter 2.3.1 --- Sample preparation for SDS-PAGE --- p.34 / Chapter 2.3.2 --- SDS-PAGE --- p.35 / Chapter 2.4 --- Immunodetection --- p.36 / Chapter 2.4.1 --- Electroblotting --- p.36 / Chapter 2.4.2 --- Western blotting --- p.36 / Chapter 2.4.3 --- Filter trap assay --- p.37 / Chapter 2.5 --- Sav antibody production --- p.38 / Chapter 2.5.1 --- Sav peptide synthesis --- p.38 / Chapter 2.5.2 --- Rabbit immunization --- p.38 / Chapter 2.6 --- Cryosectioning and immunostaining of adult fly heads --- p.39 / Chapter 2.7 --- Alcohol dehydrogenase assay --- p.40 / Chapter 2.8 --- Semi-quantitative reverse transcription- Polymerase Chain Reaction --- p.41 / Chapter 2.8.1 --- Total RNA preparation from fly heads --- p.41 / Chapter 2.8.2 --- Reverse transcription- Polymerase Chain Reaction (RT-PCR) --- p.41 / Chapter 2.9 --- Reagents and buffers --- p.42 / Chapter 3. --- RESULTS / Chapter 3.1 --- Transgenic polyglutamine fly models --- p.48 / Chapter 3.1.1 --- Characteristics of MJD polyglutamine fly model --- p.48 / Chapter 3.1.1.1 --- Overexpression of expanded truncated human MJD proteins in Drosophila causes eye degeneration --- p.49 / Chapter 3.1.1.2 --- Overexpression of expanded truncated human MJD proteins in Drosophila results in nuclear inclusion formation --- p.49 / Chapter 3.1.1.3 --- Formic acid dissolves fly polyglutamine nuclear inclusions --- p.51 / Chapter 3.1.1.3.1 --- Formic acid dissolves fly polyglutamine NIs as shown by Western blot analysis --- p.51 / Chapter 3.1.1.3.2 --- Formic acid dissolves fly polyglutamine NIs as shown by filter trap assay --- p.53 / Chapter 3.1.2 --- Summary --- p.55 / Chapter 3.2 --- Proteomic identification of nuclear inclusion (NI) proteins --- p.56 / Chapter 3.2.1 --- Proteomic identification of NI proteins by SDS-insolubility of NIs --- p.56 / Chapter 3.2.2 --- Proteomic identification of NI proteins by FA-solubility of NIs --- p.63 / Chapter 3.2.2.1 --- Two-dimensional gels showing differential protein spots as potential NI proteins --- p.63 / Chapter 3.2.2.2 --- NI protein candidates identified by the 2D approach --- p.75 / Chapter 3.2.3 --- Study of polyglutamine NI proteins by flow cytometry analysis --- p.90 / Chapter 3.2.3.1 --- Detection of fly polyglutamine NIs by flow cytometry --- p.90 / Chapter 3.2.3.2 --- Characterization of a new GFP-polyglutamine fly model --- p.92 / Chapter 3.3 --- Characterization of the nuclear inclusion protein candidates --- p.96 / Chapter 3.3.1 --- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) --- p.96 / Chapter 3.3.1.1 --- Confirmation of GAPDH as a NI protein --- p.97 / Chapter 3.3.1.2 --- Discussion --- p.97 / Chapter 3.3.2 --- Receptor of activated protein kinase C (RACK1) --- p.99 / Chapter 3.3.2.1 --- Confirmation of RACK1 as a NI protein --- p.99 / Chapter 3.3.2.1.1 --- Colocalization of RACK1 with NIs --- p.99 / Chapter 3.3.2.1.2 --- Formic Acid extracts RACK1 from NIs --- p.101 / Chapter 3.3.2.2 --- Reduction of soluble RACK1 protein level in polyglutamine fly --- p.101 / Chapter 3.3.2.2.1 --- Soluble RACK1 protein level reduced in polyglutamine fly --- p.101 / Chapter 3.3.2.2.2 --- RACK1 transcript level remains unchanged in polyglutamine fly --- p.103 / Chapter 3.3.2.3 --- Overexpression of RACK 1 partially suppresses polyglutamine degeneration --- p.105 / Chapter 3.3.2.4 --- Discussion --- p.107 / Chapter 3.3.3 --- Warts (Wts) --- p.111 / Chapter 3.3.3.1 --- Overexpression of Wts partially suppresses polyglutamine degeneration --- p.111 / Chapter 3.3.3.2 --- Wts mutant slightly enhances polyglutamine degeneration --- p.113 / Chapter 3.3.3.3 --- Genetic analysis of Warts pathway in polyglutamine pathogenesis --- p.113 / Chapter 3.3.3.3.1 --- Overexpression of Salvador partially suppresses polyglutamine degeneration --- p.116 / Chapter 3.3.3.3.2 --- Hpo mutant slightly enhances polyglutamine degeneration --- p.119 / Chapter 3.3.3.3.3 --- Overexpression of DIAP1 partially suppresses polyglutamine degeneration --- p.119 / Chapter 3.3.3.4 --- Discussion --- p.121 / Chapter 3.3.4 --- Alcohol dehydrogenase (Adh) --- p.122 / Chapter 3.3.4.1 --- Adh activity is reduced in polyglutamine flies --- p.122 / Chapter 3.3.4.2 --- Overexpression of Hsp70 partially restores the reduced Adh activity in polyglutamine flies --- p.122 / Chapter 3.3.4.3 --- Discussion --- p.125 / Chapter 3.3.5 --- Genetic analysis of other NI protein candidates --- p.127 / Chapter 3.3.5.1 --- Overexpression of CG7920 protein partially suppresses polyglutamine degeneration --- p.127 / Chapter 3.3.5.2 --- Pten dsRNA slightly enhances polyglutamine degeneration --- p.129 / Chapter 3.3.6 --- Summary --- p.131 / Chapter 4. --- DISSCUSSION / Chapter 4.1 --- Protein depletion theory --- p.133 / Chapter 4.2 --- Comparison of different approaches for identification of NI proteins --- p.134 / Chapter 4.3 --- Long-term significance --- p.136 / Chapter 4.4 --- Future studies --- p.137 / Chapter 4.4.1 --- Characterization of other NI protein candidates --- p.137 / Chapter 4.4.2 --- Study of NI proteins by an alternative approach --- p.137 / Chapter 4.4.3 --- Study of NI proteins using other polyglutamine fly models --- p.137 / Chapter 5. --- CONCLUSION --- p.139 / Chapter 6. --- REFERENCES --- p.140

Introdução: A doença por grãos argirofílicos (DGA) é uma tauopatia esporádica distinta, bastante frequente, com uma prevalência atingindo 31,3% em centenários, porém pouco reconhecida A manifestação clínica mais comum da DGA é de um comprometimento cognitivo de lenta evolução associado a uma alta frequência de sintomas psiquiátricos. O diagnóstico de DGA é possível somente através da análise do encéfalo post-mortem com os achados das três principais alterações patológicas: grãos argirofílicos, corpúsculos em embrião e pré-emaranhados neuronais. O presente estudo investigou as características demográficas, clínicas e neuropatológicas dos indivíduos com DGA e possíveis associações clínico-patológicas. Métodos: Foram estudados 983 casos (acima de 50 anos de idade) provenientes da amostra do Banco de Encéfalos do Grupo de Estudos em Envelhecimento Cerebral. A avaliação clínica e funcional foi realizada através de uma ampla entrevista semiestruturada respondida por um informante com contato próximo com o paciente. Os participantes foram estratificados conforme a presença de comprometimento cognitivo (de acordo com Escala de Avaliação Clinica da Demência) e, posteriormente, pela presença de DGA em quatro grupos: DGA com e sem comprometimento cognitivo e não-DGA com e sem comprometimento cognitivo. Análise descritiva foi realizada para dados socioeconômicos, genótipo de APOE e variáveis clínico-funcionais, neuropsiquiátricas e neuropatológicas na amostra DGA e em cada grupo. Foi utilizado um modelo de regressão logística multivariada para investigar as associações entre perfil cognitivo e sintomas neuropsiquiátricos com DGA. Resultados: DGA foi identificada em 150 indivíduos (15,1%). Idade avançada e baixo nível socioeconômico foram associados com DGA independente da presença de comprometimento cognitivo. A presença de DGA foi associada a uma redução de 60% na probabilidade de um escore >= 3.8 no Questionário do informante sobre o Declínio Cognitivo do Idoso (OR=0,40; IC de 95% 0,22-0,74; p=0,004). Adicionalmente, o subitem apetite do inventário neuropsiquiátrico foi associado à DGA em indivíduos cognitivamente normais (OR=1,85; IC de 95% 1,09-3,12; p=0,02). Conclusão: A DGA pode preservar a cognição em indivíduos com patologias neurodegenerativas associadas em particular nos casos com patologia tipo Alzheimer concomitante. A investigação dos mecanismos subjacentes a esse efeito pode auxiliar no desenvolvimento de novos tratamentos para a Doença de Alzheimer / Background: Argyrophilic grain disease (AGD) is an underrecognized, distinct, highly frequent sporadic tauopathy, with prevalence reaching 31.3% in centenarians. The most common presentation of AGD is a slowly progressive amnestic mild cognitive impairment, accompanied by high frequency of neuropsychiatric symptoms. AGD can only be diagnosed postmortem by the finding of its three main pathologic features: argyrophilic grains, oligodendrocytic coiled bodies and neuronal pretangles. The present study investigated demographic, clinical, and neuropathological profiles and analyzed clinicopathological associations. Methods: We studied 983 participants (over 50 years of age) from the Brain Bank of the Brazilian Aging Brain study group sample. Clinical and functional evaluation included demographics and a semi-structured interview covering various cognitive domains conducted with a knowledgeable informant. Participants were stratified by cognitive status (based on Clinical Dementia Rating scale), followed by the presence of AGD in four groups: AGD with and without cognitive impairmet, and non-AGD with and without cognitive impairment. Descriptive statistics were used for sociodemographic data, APOE genotypes, and the clinical, cognitive, neuropsychiatric, functional, and neuropathological variables in AGD samples and in each group. We used multivariate logistic regression models to investigate the association between the cognitive status and neuropsychiatric symptoms with AGD. Results: AGD was identified in 150 participants (15.1%). Older age and lower socioeconomic status were associated with AGD independent of cognitive status. Multivariate analyses revealed that AGD was associated with a 60% reduction in the odds of having an IQCODE >= 3.8 (OR = 0.40, 95% CI 0.22-0.74, p = 0.004) and that the NPI sub-item \"appetite and eating abnormalities\" was associated with AGD in controls (OR = 1.85, 95% CI 1.09-3.12, p = 0.02). Conclusion: AGD might preserve cognition in individuals with coexistent neurodegenerative pathologies, in particular those of the Alzheimer-type. Investigating whether the mechanisms underlying this effect could provide novel therapeutic approaches to the treatment of Alzheimer´s disease

A principal forma de tratamento da doença de Alzheimer é através de inibidores sintéticos de acetilcolinesterase. Os inibidores existentes no mercado além, de terem um custo elevado e apresentarem efeitos colaterais não previnem ou curam a doença de Alzheimer, apenas retardam o seu desenvolvimento. A baixa efetividade dos tratamentos existentes e a ausência de prognóstico positivo impulsionaram no mercado uma demanda por novas formas de tratamento e/ou prevenção da doença. Neste cenário, investigar a atividade anticolinesterásica em pimentas do reino e pimenta rosa, bem como seu potencial antioxidante e composição fitoquímica são maneiras de promover tanto o conhecimento sobre estas especiarias como beneficiar o panorama de saúde pública, seja no combate a doença de Alzheimer e/ou outras pandemias existentes. Com o objetivo de avaliar o potencial destas especiarias, extratos metanólicos de pimenta preta, verde, branca e rosa foram avaliados segundo o teor de fenólicos totais, atividade antioxidante pelo método DPPH, ABTS e FRAP e atividade anticolinesterásica. Posteriormente o perfil metabolômico dos grãos e extratos de pimenta foi analisado por cromatografia líquida acoplada à espectrometria de massa. As amostras foram ionizadas por -eletrospray? operando em modo negativo e positivo. O extrato de pimenta rosa apresentou o maior teor de compostos fenólicos totais e a melhor atividade antioxidante e anticolinesterásica em comparação aos demais extratos. Dentre as pimentas do reino, a pimenta verde destacou-se com os maiores teores de compostos fenólicos e atividade antioxidante pelo método DPPH e ABTS. Já a pimenta preta mostrou os melhores resultados para as análises de atividade anticolinesterásica e atividade antioxidante pelo método FRAP. Quanto ao perfil metabolômico as amostras de pimenta foram significativamente distintas entre si. Os espectros obtidos para as amostras de grãos de pimenta foram mais ricos em metabólitos em relação às amostras de extrato em ambos os modos operacionais. O processamento das pimentas do reino induziu modificações quantitativas e qualitativas sobre a composição fitoquímica deste condimento. A pimenta rosa não apresentou nenhum possível metabólito com alto potencial discriminatório, dentre os 15 íons investigados. Em modo operacional negativo, os metabólitos presentes no extrato de pimenta verde, exceto íon de m/z 329,2081, foram responsáveis por todas as contribuições mais significativas entre os perfis metabolômicos gerados. A partir dos resultados obtidos no presente trabalho é possível afirmar que a pimenta rosa apresenta o maior potencial na redução de risco da doença de Alzheimer, dentre as pimentas investigadas. / Synthetic inhibitors of cholinesterase are the main drugs used in the treatment for Alzheimer\'s disease and besides the high cost and side effects, the current drugs available in the market do not prevent or cure Alzheimer, only retard its development. The absence of positive prognosis and limited effectiveness of existing drugs enhance the demand for new forms of the disease\'s treatment and/or prevention. In this scenario, investigating Piper nigrum and Schinus terebinthifolius peppercorns for its acetylcholinesterase and antioxidant activity, as well as its phytochemical composition, are ways to promote both knowledge about these spices and the perspective of health, either in combat of Alzheimer\'s disease and/or other existing pandemics. In order to evaluate the potential of these spices, black, green, white and pink peppers methanolic extracts were evaluated according to the total phenolic content, antioxidant activity by DPPH, ABTS and FRAP assay and anticholinesterase activity. Subsequently the metabolomic profile of the extracts and peppercorns were analyzed by mass spectrometry coupled to liquid chromatography. Samples were ionized by electrospray operating in negative and positive modes. The pink pepper (Schinus terebinthifolius) extract showed the highest content of total phenolic compounds and the best antioxidant and anticholinesterase activity compared to the other extracts. Among Piper nigrum peppercorns, green pepper stood out as the highest levels of phenolic compounds and antioxidant activity by DPPH and ABTS methods. Black pepper obtained the best results for the analysis of acetylcholinesterase activity and antioxidant activity by FRAP method. Regarding the metabolomic profiles, pepper samples are significantly different from each other. The spectrum obtained for peppercorn\'s samples were richer in metabolites compared to extract\'s samples in both operating modes. The processing induced quantitative and qualitative changes on the phytochemical composition of the peppercorns. The pink pepper did not show any possible metabolite with high discriminatory potential, of the 15 ions investigated. In the negative operating mode, the metabolites present in the extracts of green pepper, excluding m/z 329.2081 ion, were responsible for all of the most significant contributions between metabolomic profiles generated. From the results obtained in this study it is possible to affirm that pink pepper has the greatest potential to reduce Alzheimer\'s disease risk, among peppers investigated.

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>