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Objective: This study aimed to investigate the role and prognosis of Alzheimer disease biomarkers in patients with mild cognitive impairment (MCI) at a memory clinic in Latin America. Methods: We studied 89 patients with MCI, 43 with Alzheimer-type dementia, and 18 healthy controls (matched for age, sex, and educational level) at our memory clinic (Instituto FLENI) in Buenos Aires, Argentina. Patients and controls underwent an extensive demographic, neurological, and neuropsychological assessment. All subjects underwent a brain MRI scan; FDG-PET scan; amyloid PET scan; apolipoprotein E genotyping; and cerebrospinal fluid concentrations of Aβ1-42, tau, and phosphorylated tau. Patients were categorised as positive or negative for the presence of amyloid pathology and neurodegeneration. Results: Amyloid pathology was observed in cerebrospinal fluid results in 18% of controls, 64% of patients with MCI, and 92% of patients with Alzheimer-type dementia. Suspected non–Alzheimer disease pathophysiology was found in 11% of controls, 6% of patients with MCI, and 8% of patients with Alzheimer-type dementia. At 30 months of follow-up, 45% of amyloid-positive patients with MCI and 20% of amyloid-negative patients with MCI showed progression to dementia. Conclusions: This study demonstrates biomarker-based MCI prognosis and supports its role in clinical decision-making in daily practice. Objetivo: este estudio tuvo como objetivo investigar el papel y el pronóstico de los biomarcadores de la enfermedad de Alzheimer en pacientes con deterioro cognitivo leve (MCI) en una clínica de memoria en América Latina. Métodos: Estudiamos 89 pacientes con LM, 43 con demencia de tipo Alzheimer y 18 controles sanos (emparejados por edad, sexo y nivel educativo) en nuestra clínica de memoria (Instituto FLENI) en Buenos Aires, Argentina. Los pacientes y los controles se sometieron a una extensa evaluación demográfica, neurológica y neuropsicológica. Todos los sujetos se sometieron a una resonancia magnética cerebral; FDG-PET scan; tomografía PET amiloide; apolipoproteína E genotipado; y concentraciones de líquido cefalorraquídeo de Aβ1-42, tau y tau fosforilada. Los pacientes fueron categorizados como positivos o negativos por la presencia de patología amiloide y neurodegeneración. Resultados: se observó patología de amiloide en los resultados del líquido cefalorraquídeo en el 18% de los controles, el 64% de los pacientes con LM y el 92% de los pacientes con demencia de tipo Alzheimer. La sospecha de fisiopatología no relacionada con la enfermedad de Alzheimer se encontró en el 11% de los controles, el 6% de los pacientes con MCI y el 8% de los pacientes con demencia de tipo Alzheimer. A los 30 meses de seguimiento, el 45% de los pacientes con amiloide positivo con MCI y el 20% de los pacientes con amiloide negativo con MCI mostraron progresión a demencia. Conclusiones: este estudio demuestra el pronóstico de MCI basado en biomarcadores y apoya su papel en la toma de decisiones clínicas en la práctica diaria.

Neurodegenerative dementias are characterized by the abnormal accumulation of misfolded proteins. However, its diagnostic criteria are still based on the clinical phenotype. The development of biomarkers allowed in vivo detection of pathophysiological processes. This article aims to make a non-systematic review of the use of molecular neuroimaging as a biomarker. Molecular neuroimaging is based on the use of radiotracers for image acquisition. The radiotracer most used in PET is 18F-fluorodeoxyglucose (FDG), with which it is possible to study the regional brain glucose metabolism. The pattern of regional hypometabolism provides neuroanatomical information on the neurodegenerative process, which, in turn, has a good specificity for each type of proteinopathy. FDG is very useful in the differential diagnosis of neurodegenerative dementias through the regional pattern of involvement, including dementia with Lewy bodies and the spectrum of frontotemporal dementia. More recently, radiotracers with specific ligands to some of the pathological proteins have been developed. Pittsburgh compound B (PIB) labeled with 11C and the ligands that use 18F (florbetapir, florbetaben and flutemetamol) are the most used radiotracers for the detection of insoluble β-amyloid peptide in Alzheimer's disease (AD). A first generation of ligands for tau protein has been developed, but it has some affinity for other non-tau protein aggregates. A second generation has the advantage of having a higher affinity for hyperphosphorylated tau protein, including in primary tauopathies. RESUMO As demências neurodegenerativas caracterizam-se pelo acúmulo anormal de proteínas mal dobradas. Entretanto, os seus critérios diagnósticos ainda se baseiam no fenótipo clínico. O desenvolvimento de biomarcadores permitiu a detecção in vivo do processo fisiopatológico. O objetivo deste artigo é fazer uma revisão não-sistemática sobre o papel da neuroimagem molecular como biomarcador. A neuroimagem molecular baseia-se no uso de radiotraçadores para aquisição da imagem. O mais usado no PET é o 18F-fluorodeoxiglicose (FDG), com o qual é possível estudar o metabolismo regional de glicose cerebral. O padrão de hipometabolismo regional fornece uma informação neuroanatômica do processo neurodegenerativo que, por sua vez, tem uma boa especificidade para cada tipo de proteinopatia. O PET-FDG é muito útil no diagnóstico diferencial das demências neurodegenerativas através do padrão de acometimento regional, incluindo a demência com corpos de Lewy e o espectro das demências frontotemporais. Mais recentemente, radiotraçadores com ligantes específicos a algumas das proteínas patológicas têm sido desenvolvidos. O composto B de Pittsburgh (PIB) com 11C e os ligantes dos que usam 18F (florbetapir, florbetaben e flutemetamol) são os radiotraçadores mais usados para a detecção de peptídeo β-amiloide insolúvel na doença de Alzheimer (DA). Uma primeira geração de ligantes para proteína tau foi desenvolvida, mas apresenta alguma afinidade a outros agregados proteicos não-tau. Uma segunda geração tem a vantagem de apresentar uma maior afinidade à proteína tau hiperfosforilada, incluindo nas taupatias primárias.

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive and irreversible loss of cognitive function. The presence of senile plaques is one of the pathological markers of the disease and is associated with the onset of neuroinflammatory mechanisms. The exact pathophysiology of AD has not been completely understood, and there are no curative therapies yet. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenol that is noted for its antioxidant and anti-inflammatory properties. Objective: To review the role of resveratrol in the pathophysiological aspects of AD. Methods: This study carried out a literature review using PubMed/Medline, Virtual Health Library (VHL), Web of Sciences, SCOPUS and the Cochrane Library databases. Original research articles, describing both in vitro and in vivo experiments, published between 2008 and 2018, were included. Results: We identified 667 articles, of which 619 were excluded because they were repeated or did not follow the inclusion criteria. The present study includes the remaining 48 articles. Discussion: Resveratrol demonstrates beneficial and protective effects in AD models and seems to provide a promising therapeutic alternative. Conclusion: Although resveratrol appears to mitigate some pathophysiological aspects of AD, further studies are needed to prove the safety and efficacy of this compound in humans. RESUMO Introdução: A doença de Alzheimer (DA) é neurodegenerativa e caracterizada por perda progressiva e irreversível da função cognitiva. A presença de placas senis é um dos marcadores patológicos da doença e está associada ao aparecimento de mecanismos neuroinflamatórios. A fisiopatologia exata da DA ainda não é completamente compreendida, e ainda não existem terapias curativas. O resveratrol (3,5,4'-trihidroxi-trans-estilbeno) é um polifenol conhecido por suas propriedades antioxidantes e anti-inflamatórias. Objetivo: Revisar o papel do resveratrol nos aspectos fisiopatológicos da DA. Métodos: Este estudo realizou uma revisão narrativa da literatura a partir das bases de dados PubMed/Medline, Biblioteca Virtual em Saúde (BVS), Web of Science, SCOPUS e Cochrane Library. Foram incluídos artigos originais, realizados in vitro e in vivo, publicados entre 2008 e 2018. Resultados: Foram identificados 667 artigos, dos quais 619 foram excluídos por estarem repetidos ou não se enquadrarem nos critérios de inclusão. O presente estudo inclui os 48 artigos restantes. Discussão: O resveratrol demonstra efeitos benéficos e protetores em modelos de DA, bem como parece fornecer uma alternativa terapêutica promissora. Conclusão: Embora o resveratrol pareça atenuar alguns aspectos fisiopatológicos da DA, são necessários mais estudos para comprovar a segurança e a eficácia deste composto em seres humanos.

25 p. : il. -- Bibliogr.: p. 22-25 [ES] Recientes investigaciones han demostrado que el malplegamiento proteico es la principal causa subyacente de las enfermedades neurodegenerativas más prevalentes. Hasta la fecha, el mayor conocimiento recopilado sobre las bases moleculares del malplegamiento proteico, lo recogen las investigaciones acerca de la proteína priónica, una glicoproteína de membrana con tendencia al malplegamiento. En condiciones específicas, dicha proteína puede adquirir una conformación aberrante de naturaleza amiloide, causante de un grupo de patologías llamadas encefalopatías espongiformes transmisibles. El desarrollo de estas patologías implica largos periodos de incubación, pero una vez comienzan los signos clínicos, la resolución es fatal en todos los casos. En este trabajo de fin de grado se han aportado nuevos datos para un tipo de estrategia terapéutica basada en proteínas priónicas recombinantes dominantes negativas. Es decir, proteínas priónicas que, debido a mutaciones singulares, presentan resistencia al malplegamiento e inhiben el mismo en proteínas priónicas susceptibles. Mediante el uso de la amplificación por agitación del malplegamiento proteico, una técnica in vitro de generación de priones, se evaluó la tendencia al malplegamiento de la proteína priónica de Myodes glareolus con siete mutaciones basadas en prolina: T107P, R136P, R164P, N170P, T191P, Q212P y E227P. Ninguna mutación resultó en proteínas resistentes al malplegamiento espontáneo. Aun así, la proteína priónica que poseía la mutación R164P resistió el malplegamiento inducido por seis cepas priónicas distintas, indicando una posible capacidad dominante negativa. [EN] Recent research has confirmed that protein misfolding is the principal cause underlying the most prevalent neurodegenerative diseases. To date the greatest knowledge gathered about the molecular basis of protein misfolding is on the prion protein, a misfolding prone cell-surface glycoprotein. Under certain conditions, the latter protein can acquire an aberrant amyloid conformation that causes a group of pathologies known as transmissible spongiform encephalopathies. The development of these pathologies implies long incubation periods but, once clinical signs arise, the resolution is in all cases fatal. This final degree thesis has provided new evidence for a therapeutic strategy based on dominant negative recombinant prion proteins, that is, prion proteins which, due to singular mutations, are able to resist protein misfolding and are able to inhibit the misfolding of other susceptible prion proteins. Through the use of protein misfolding shaking amplification, a prion generating in vitro technique, the misfolding proneness of seven proline-based mutated Myodes glareolus prion proteins was evaluated: T107P, R136P, R164P, N170P, T191P, Q212P and E227P. No mutation produced spontaneous misfolding resistant proteins. Even so, the prion protein that possessed the R164P mutation withstood induced misfolding by six different prion strains, indicating possible dominant negative properties.

Las proteínas amiloides tienen la tendencia natural de formar fibras insolubles a partir de monómeros solubles, un proceso que implica la formación de agregados de distintos tamaños, y con distintas conformaciones. Una de características definitorias de las fibras amiloides es el enriquecimiento en láminas beta que confieren propiedades tintoriales, bioquímicas y biológicas. A pesar de preservar la denominación patológica, los amiloides median funciones que son esenciales para la homeostasis celular o la adaptación al entorno. En Bacillus subtilis, la proteína amiloide TasA desempeña varias de estas funciones: i) estructural, contribuyendo al ensamblaje de la matriz extracelular, ii) celular, preservando la fluidez y funcionalidad de la membrana, iii) señalización, manteniendo ciertas subpoblaciones celulares. Por lo tanto, resulta esencial conocer los residuos o dominios de la proteína que hacen posible a TasA transitar entre las distintas conformaciones, como paso previo a entender esta diversidad de funciones. En trabajos previos (1), estudiamos las propiedades estructurales de TasA en su forma fibrilar, y mediante la digestión con proteínasa K demostramos la existencia de una región resistente en la mitad amino terminal y que denominamos el núcleo amiloide. En este trabajo se ha estudiado esta región mediante el uso de diversas aproximaciones, genética, bioinformáticas, bioquímicas o biofísicas. Como resultado hemos detectado la existencia de residuos aminoácidos y regiones conservadas que parecen ser esenciales al menos en el proceso de amiloidogenesis, y por tanto en el papel estructural involucrado en la formación de biofilm. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

En este proyecto se estudian las distintas metodologías de pre-procesado de imágenes tau-PET que cuantifican la cantidad y distribución de proteína tau dentro del cerebro. Se estudian los métodos de registro para agregarlas a imágenes de RM que se usan en la praxis usual dentro del campo de la neuroimagen así como técnicas innovadores planteadas dentro del grupo de investigación de la Unidad de Memoria del Hospital Sant Pau. Finalmente, se proponen modelos de regresión lineal para estudiar las capacidades clasificativas y predictivas de la medida de SUVr para el diagnostico de posibles pacientes. In this project, some of the most used pre-processing techniques for tau-PET imaging are studied. This imaging technique provides information on the quantity as well as topographical distribution of tau protein throughout the brain structure. Furthermore, we characterize the state-of-the-art registration process configurations, as well as novel techniques developed inside the Sant Pau Hospital Memory Unit, in order to align tau-PET images to MRI scans. Finally, we propose different logistic regression models in order to assess the classification and prediction capabilities of the SUVr measure for the diagnosis of potential patients.

Les malalties neurodegeneratives, com el Parkinson o l'Alzheimer, són un dels reptes més grans de la societat moderna. La manca de tractaments efectius i tècniques de diagnòstic precoç, juntament amb l'envelliment gradual de la població, contribueix a agreujar aquesta situació, impactant significativament en la societat i els sistemes de salut. Per tant, desenvolupar noves tècniques diagnòstiques i terapèutiques és un objectiu imperatiu. Nombrosos trastorns neurodegeneratius es distingeixen per la presència d'acumulacions proteiques intra/extracel·lulars com a conseqüència de la formació d'agregats molt ordenats anomenats amiloides, que presenten una gran toxicitat i capacitat de transmissió. En particular, la malaltia de Parkinson es caracteritza per la pèrdua de cèl·lules dopaminèrgiques a la substància negra pars compacta el que indueix un important dèficit motor que provoca bradicinèsia, tremolor o rigidesa. Tot i que es desconeix la causa de la malaltia de Parkinson, els teixits afectats presenten acumulacions intracel·lulars de proteïnes, els components principals de les quals són les estructures amiloides de l'α-Sinucleïna. Aquesta proteïna, caracteritzada per la seva naturalesa desordenada, es localitza preferentment als extrems presinàptics com a monòmer soluble. Tot i això, en condicions patològiques, l'α-Sinucleïna estableix interaccions intermoleculars que condueixen a la formació de fibrilles amiloides insolubles. Aquests agregats fibril·lars alteren significativament l'homeòstasi cel·lular i s'acumulen als cossos de Lewy o neurites, segrestant diversos components cel·lulars al llarg del procés. A més, les estructures intermèdies formades durant l'agregació, com ara oligòmers o protofibril·les, s'han descrit com a entitats tòxiques i transmissibles que indueixen la mort cel·lular i la propagació de la patologia de cèl·lula a cèl·lula. Malauradament, la complexitat i l'heterogeneïtat d'aquest procés han dificultat el desenvolupament de teràpies i diagnòstics efectius per a la malaltia de Parkinson. Aquesta tesi pretén desenvolupar molècules efectives que impedeixin l'agregació de proteïnes, redueixin la toxicitat i la propagació de l'amiloide. En implementar una tècnica robusta per a l'anàlisi de grans biblioteques químiques, hem descobert i caracteritzat diferents xaperones moleculars potents que obstaculitzen de manera eficient l'agregació in vitro de α-Sinucleïna. A més, el treball aquí descrit aclareix el mecanisme inhibitori i les propietats de les molècules obtingudes. També aclarim les estructures o passos del procés abordat pels compostos, la concentració mínima efectiva i el seu efecte en variants familiars de la malaltia de Parkinson. A més, com que l'α-Sinucleïna pot adoptar múltiples conformacions amiloides depenent de les condicions ambientals, aquesta tesi també se centra a discernir si la capacitat inhibidora d'aquestes molècules és consistent davant de diferents ceps amiloides. En conjunt, l'objectiu final d'aquest treball és identificar validar i caracteritzar els mecanismes d'acció de diferents estructures moleculars, que poden evolucionar potencialment a fàrmacs modificats de la malaltia, presentant una activitat neuroprotectora Las enfermedades neurodegenerativas, como el Parkinson o el Alzheimer, son uno de los mayores desafíos de la sociedad moderna. La falta de tratamientos efectivos y técnicas de diagnóstico precoz, junto con el paulatino envejecimiento de la población, contribuye a agravar esta situación, impactando significativamente en la sociedad y los sistemas de salud. Por lo tanto, desarrollar nuevas técnicas diagnósticas y terapéuticas es un objetivo imperativo. Numerosos trastornos neurodegenerativos se distinguen por la presencia de acumulaciones proteicas intra/extracelulares como consecuencia de la formación de agregados muy ordenados denominados amiloides, que presentan una gran toxicidad y capacidad de transmisión. En particular, la enfermedad de Parkinson se caracteriza por la pérdida de células dopaminérgicas en la sustancia negra pars compacta lo que induce un importante déficit motor que provoca bradicinesia, temblor o rigidez. A pesar de que se desconoce la causa de la enfermedad de Parkinson, los tejidos afectados presentan acumulaciones intracelulares de proteínas, cuyo principal componente son las estructuras amiloides de la α-Sinucleína. Esta proteína, caracterizada por su naturaleza desordenada, se localiza preferentemente en los extremos presinápticos como monómero soluble. Sin embargo, en condiciones patológicas, la α-Sinucleína establece interacciones intermoleculares que conducen a la formación de fibrillas amiloides insolubles. Estos agregados fibrilares alteran significativamente la homeostasis celular y se acumulan en los cuerpos de Lewy o neuritas, secuestrando varios componentes celulares a lo largo del proceso. Además, las estructuras intermedias formadas durante la agregación, como oligómeros o protofibrillas, se han descrito como entidades tóxicas y transmisibles que inducen la muerte celular y la propagación de la patología de célula a célula. Desafortunadamente, la complejidad y heterogeneidad de este proceso han dificultado el desarrollo de terapias y diagnósticos efectivos para la enfermedad de Parkinson. La presente tesis pretende desarrollar moléculas efectivas que impidan la agregación de proteínas, reduciendo la toxicidad y propagación del amiloide. Al implementar una técnica robusta para el análisis de grandes bibliotecas químicas, hemos descubierto y caracterizado diferentes chaperonas moleculares potentes que detienen de manera eficiente la agregación in vitro de α-Sinucleína. Además, el trabajo aquí descrito aclara el mecanismo inhibitorio y las propiedades de las moléculas obtenidas. También aclaramos las estructuras o pasos del proceso abordados por los compuestos, su concentración mínima efectiva y su efecto en variantes familiares de la enfermedad de Parkinson. Además, como la α-Sinucleína puede adoptar múltiples conformaciones amiloides dependiendo de las condiciones ambientales, esta tesis también se centra en discernir si la capacidad inhibidora de estas moléculas es consistente frente a diferentes cepas amiloides. En conjunto, el objetivo final de este trabajo es identificar validar y caracterizar los mecanismos de acción de diferentes estructuras moleculares, que pueden evolucionar potencialmente a fármacos modificadores de la enfermedad, presentando una actividad neuroprotectora. Neurodegenerative disorders, such as Parkinson's or Alzheimer's diseases, are one of the biggest health challenges in modern society. The lack of effective treatments and early-diagnosis techniques, together with the gradual aging population, contributes to worsening these conditions, significantly impacting society and the healthcare system. Therefore, developing new diagnostic and therapeutic techniques is a necessary goal. Many neurodegenerative disorders are characterised by the presence of intra/extracellular proteinaceous accumulations, often because of the formation of highly ordered aggregates named amyloids, which exhibit toxicity and transmission capacities. In particular, Parkinson's disease is characterised by the loss of dopaminergic cells in substantia nigra pars compacta, which induces motor deficits, causing bradykinesia, tremor or rigidity. Although the cause of Parkinson's disease remains unknown, affected tissues exhibit intracellular accumulations of proteins, whose main components are amyloid forms of α-Synuclein. This protein, characterised by its disordered nature, is preferentially located at the presynaptic termini as a soluble monomer. Nevertheless, in pathological conditions, α-Synuclein establishes intermolecular interactions that lead to the formation of insoluble amyloids fibrils. These fibrillar aggregates alter cellular homeostasis and accumulate into Lewy's bodies or neurites, kidnaping several cellular components along the process. Moreover, intermediates formed during aggregation, as oligomers or protofibrils, have been described as toxic and transmissible entities that induce cell death and cell-to-cell spreading of the pathology. Unfortunately, the complexity and heterogeneity of this process have hindered the development of effective therapies and diagnostic tools for Parkinson's Disease. The present thesis attempts to develop effective molecules that prevent α-Synuclein aggregation, reducing amyloid toxicity and spreading. By implementing a robust technique for the screening of large chemical libraries we have discovered and characterised different molecular chaperones that efficiently halt α-Synuclein in vitro aggregation. In addition, the work here described elucidates the inhibitory mechanism and the properties of the obtained molecules. We also clarified the structures or steps of the process targeted by the compounds, their minimum effective concentration, and their effect on familiar variants of Parkinson's disease. Furthermore, as α-Synuclein can adopt multiple amyloid conformations depending on the environmental conditions, this thesis also addresses if the molecules inhibitory capacity is consistent against different amyloid strains. Altogether, the final goal of this work is to identify, validate and characterize the mechanisms of action of different molecular scaffolds that can be potentially evolved into disease-modifying drugs with neuroprotective activity.

The human pathogen Bacillus cereus is responsible of many food poisoning due to the ingestion of contaminated vegetables or processed foods. For the survival and colonization, the bacteria form a biofilm on biotic and abiotic surfaces in which a specific subpopulation differentiates to produce a multifunctional extracellular matrix (ECM), mainly composed by exopolysaccharides, proteins and extracellular DNA. One of the most attractive components of the ECM are the amyloid proteins due to its high tendency to fibrillate and the multifunctional role in the bacterial physiology. In the B. cereus genome there is a specific genomic region that contains two orthologues genes to the tasA gene in Bacillus subtilis, initially described as essential in the assembly of amyloid fibers, named tasA and calY respectively. The deletion of these genes gives a different phenotype related with a deficient biofilm formation in both cases. In the tasA mutant, the biofilm finally detaches at 72 hours, and the calY mutant shows a thinner ring phenotype in comparison with the wild type strain. The gene expression dynamic during the biofilm development reveals that there are three different subpopulations: both genes are expressed, only calY or none of them. Furthermore, transcriptomic analysis by RNA-Seq show that the mutants have different changes in the gene regulation and the calY mutant is the most affected, with 227 genes deregulated that affect to several important pathways such as the synthesis of extracellular DNA, an important component of the ECM. The results mentioned above indicate that TasA and CalY might have complementary roles in the biopellicle development but also evidence that this physiological process is more complex in B. cereus in comparison with the model described in B. subtilis. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Beneficial microbes are known to stimulate the germination of the seeds; however, the exact mechanisms mediating these interactions are only beginning. Bacillus subtilis is a commonly detected member of the plant holobiont and provides multifaceted traits to the plant health. In this work, we demonstrated that B. subtilis triggered genetic and physiological responses in seeds that resulted in changes in the metabolic and developmental status of adult plants. A multidisciplinary approach based on microscopy, transcriptomics and metabolomics demonstrated that the chemically diverse extracellular matrix of Bacillus structurally cooperate in bacterial colonization of the seed storage tissues. The amyloid protein TasA and fengycin, two components of the extracellular matrix, differentially stimulated levels of ROS inside seeds after imbibition and targeted the oil bodies of the seed endosperm, provoking specific changes in lipid metabolism or accumulation of glutathione-related molecules that resulted in two different plant growth programs: the development of seed radicles or major growth and immunization of adult plants. Our findings prove the versatility of the bacterial ECM in establishing a mutualistic interaction with plants. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Orientador: Marisa Masumi Beppu Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química Resumo: A doença de Alzheimer está relacionada à ligação anômala que ocorre entre o peptídeo ?- amilóide (?A) e o íon cobre. Segundo a literatura, utilizando-se espectroscopia Raman, observou-se forte evidência de que o cobre se liga ao núcleo da ?A através de anéis imidazólicos de histidina. Agentes quelantes, em princípio, podem ser usados farmacologicamente no tratamento da intoxicação com metais pesados. Neste trabalho, verificou-se a ação quelante da quitosana atuando na remoção de íons cobre, na presença de ?-amilóide ou de um composto equivalente que contenha histidinas (conhecidos como os responsáveis pela ligação com o íon cobre). Devido a limitações no uso da ?A, decidiu-se por usar histidina, para um melhor entendimento da interação que ocorre no sistema. Inicialmente, esferas porosas de quitosana foram preparadas para utilização na adsorção de íons cobre, com e sem a presença de histidina. Estudos de equilíbrio, envolvendo isotermas e cinéticas de adsorção foram realizadas, e a partir desses constatou-se, que a histidina compete com a quitosana pelos íons cobre, levando a uma diminuição na capacidade de adsorção. Em contrapartida, observa-se uma diminuição não tão brusca da capacidade de adsorção, mostrando que mesmo com a competição ocorrendo a quitosana continua adsorvendo cobre. Com a aplicação dos modelos isotérmicos, foi possível prever que a adsorção ocorre em monocamadas e também em sítios heterogêneos e que não existe somente um grupamento responsável pela quelação dos íons cobre. Em relação aos modelos cinéticos foi proposto que a etapa limitante no processo é adsorção química. Empregando Espectroscopia de Infravermelho por Transformada de Fourier (FTIR-ATR), observou-se que a histidina atua principalmente nos grupos amino e hidroxil da quitosana, afetando os átomos de nitrogênio e oxigênio do mesmo. Foram também realizadas análises de espalhamento de Raios-X a baixo ângulo (SAXS) revelando que a adição de histidina ou ?A no sistema proporciona provavelmente um desenovelamento da quitosana. Através de análises de Estrutura Fina Estendida de Absorção de Raios-X (EXAFS) verificou-se que após adição de histidina ocorre uma mudança qualitativa relacionada à primeira esfera de coordenação referente à ligação de Cu-O, o que pode indicar novas ligações, agora com os átomos de nitrogênio. Para finalizar, uma isoterma de adsorção empregando-se o peptídeo ?A foi obtida, os resultados foram semelhantes aos obtidos com a histidina, mantendo-se uma de adsorção de cobre por parte da quitosana Abstract: Alzheimer's disease is related to the anomalous binding that occurs between the protein ?-amyloid (?A) and copper metal ion. By using Raman spectroscopy, literature has shown strong evidence that copper and zinc bind to A? peptide core through the imidazole ring of histidine. Chelating agents, in principle, can be used pharmacologically in the treatment of heavy metal poisoning. In this study, we verified the chelating action of chitosan acting in the removal of copper ions in the presence of amyloid-ß or an equivalent compound structural that presents histidine (known as the responsible for binding with copper ion). Due to limitations in using ?A, it was decided to use just histidine, aiming a better understanding of the interaction that occurs in the system. Initially, porous chitosan beads were prepared for the use in the adsorption of copper ions, with and without the presence of histidine. Equilibrium studies involving adsorption isotherms and kinetics were carried out, and from them it was observed that histidine competes with chitosan for the copper ions, leading to a decrease in the adsorption capacity. On the other hand, there is no sharp decrease of the adsorption capacity showing that even with a competition taking place, chitosan remains adsorbing copper. By applying the isothermal models, it was possible to predict that an adsorption monolayer occurs and that there is not only one group responsible for chelation of copper ions. With regard to kinetic models it was proposed that the limiting step in the adsorption process is the chemical adsorption. By using Fourier Transform Infrared Spectroscopy (FTIR-ATR) it was observed that histidine acts mainly in the amino and hydroxyl groups of chitosan, affecting the nitrogen and oxygen atoms of the same. Analyses were performed for Small Angle X-Ray Scattering (SAXS) revealing that the addition of histidine or ?A in the system provides probably one unfolding of chitosan. Through the analyses of Extended X-Ray absorption fine structure (EXAFS), it was found that after addition of histidine a qualitative change occurs in the first coordination sphere attributed to Cu-O binding, which may indicate new bindings, now with nitrogen atoms. Finally, the adsorption isotherm employing ?A protein was similar to those obtained with histidine; chich shows that chitosan kept the ability to absorb copper Resumo: A hidrogenólise da sacarose é uma reação química de interesse industrial, uma vez que os produtos obtidos, sob a forma de glicóis e polióis, podem ser empregados em diversos setores produtivos, tais como indústrias farmacêuticas e de alimentos. Nesse contexto, o presente trabalho tem como objetivo estudar a preparação de catalisadores de Ni e Ru suportados em carvão ativado, destinados à hidrogenólise da sacarose em meio aquoso. Para tanto, catalisadores com uma fração mássica total de 5 % de metal foram preparados através dos métodos de impregnação incipiente e úmida, a partir de soluções aquosas de precursores clorados. Antes das impregnações, o suporte de carvão ativado comercial foi submetido a um tratamento químico com solução aquosa de KOH. Os sólidos preparados por meio da impregnação incipiente foram reduzidos a 473 K (200 ºC), sob fluxo de H2. Por sua vez, a impregnação úmida foi conduzida a 353 K (80 ºC), para diversos valores de pH, sendo a redução dos catalisadores realizada com formaldeído ou NaBH4. Os sólidos foram caracterizados através das técnicas de adsorção de N2 (método de B.E.T.), titulação de Boehm, titulação potenciométrica, espectroscopia de fotoelétrons excitados por raios X, microscopias eletrônicas de varredura e de transmissão, difratometria de raios X e redução à temperatura programada. Os desempenhos catalíticos na reação de hidrogenólise da sacarose foram conduzidos num reator Parr do tipo "slurry", à temperatura de 523 K (250 ºC) e sob pressão de H2 de 5,0 MPa (50 atm), utilizando-se uma massa de 5 g do catalisador. Os resultados revelam que os catalisadores de Ni/C preparados por impregnação úmida são mais ativos e seletivos a 1,2 propanodiol que os preparados via impregnação incipiente. A impregnação úmida também leva a catalisadores de Ru/C seletivos, que se mostram mais ativos que os preparados por impregnação incipiente. Contudo, a impregnação incipiente permite obter catalisadores igualmente seletivos, sendo que, em ambos os casos, o tratamento do suporte com KOH tem um efeito marcante no aumento do rendimento de 1,2 propanodiol Abstract: The sucrose hydrogenolysis is a chemical reaction of industrial interest, since the obtained products, under the form of glycols and polyols, can be employed in different production sectors such as pharmaceutical and food industries. The present work aims to study the preparation of Ni and Ru catalysts supported on activated carbon, for the sucrose hydrogenolysis in aqueous media. For this purpose, catalysts with a total fraction of 5 wt % of metal were prepared by the methods of incipient and wet impregnation from aqueous solutions of chlorinated precursors. Before impregnation, the support of activated carbon was subjected to a chemical treatment in an aqueous solution of KOH. The solids prepared by incipient impregnation were reduced at 473 K (200 °C) under H2 flow. In its turn, the wet impregnation was conducted at 353 K (80 °C), for various pH values, being the catalysts reduction performed with formaldehyde or NaBH4. The solids were characterized by the techniques of N2 adsorption (B.E.T. method), Boehm titration, potentiometric titration, X ray photoelectron spectroscopy, scanning and transmission electronic microscopies, X ray diffraction and temperature programmed reduction. The catalytic performances for the sucrose hydrogenolysis were conducted in a Parr reactor of slurry type at temperature of 523 K (250 ° C) under H2 pressure of 5.0 MPa (50 atm), employing a catalyst mass of 5 g. The results reveal that the Ni/C catalysts prepared by wet impregnation are more active and selective for 1,2 propanediol than those prepared via incipient impregnation. The wet impregnation also leads to selective Ru/C catalysts, which are more active than those prepared by incipient impregnation. However, the incipient impregnation achieves also to selective catalysts, being that in both cases, the support treatment with KOH has a marked effect on increasing the yield of 1,2 propanediol Mestrado Engenharia de Processos Mestra em Engenharia Química

Background The described relationship between Alzheimer’s disease (AD) and type 2 diabetes (T2D) and the fact that AD has no succesful treatment has led to the study of antidiabetic drugs that may limit or slow down AD pathology. Main body Although T2D treatment has evident limitations, options are increasing including glucagon-like peptide 1 analogs. Among these, liraglutide (LRGT) is commonly used by T2D patients to improve β cell function and suppress glucagon to restore normoglycaemia. Interestingly, LRGT also counterbalances altered brain metabolism and has anti-inflammatory properties. Previous studies have reported its capacity to reduce AD pathology, including amyloid production and deposition, tau hyperphosphorylation, or neuronal and synaptic loss in animal models of AD, accompanied by cognitive improvement. Given the beneficial effects of LRGT at central level, studies in patients have been carried out, showing modest beneficial effects. At present, the ELAD trial (Evaluating Liraglutide in Alzheimer’s Disease NCT01843075) is an ongoing phase IIb study in patients with mild AD. In this minireview, we resume the outcomes of LRGT treatment in preclinical models of AD as well as the available results in patients up to date. Conclusion The effects of LRGT on animal models show significant benefits in AD pathology and cognitive impairment. While studies in patients are limited, ongoing clinical trials will probably provide more definitive conclusions on the role of LRGT in AD patients. Yes

Orientador: Carlos Henrique Inácio Ramos Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Proteínas enoveladas incorretamente, com freqüência levam à formação de agregados fibrilares contendo extensas estruturas em folha-?, comumente denominadas de fibrilas amilóides. A hipótese acerca da capacidade de formar fibrilas amilóides com estruturas idênticas e ricas em estruturas beta, ser uma propriedade genérica de toda proteína, apoia-se no fato de até mesmo proteínas sem conexão com doenças, como a mioglobina, serem capazes de gerar estruturas fibrilares. Embora várias proteínas sejam intrinsecamente desordenadas, muitas são apropriadamente empacotadas, podendo se desenovelar totalmente ou parcialmente de maneira a expor regiões propensas à agregação, que podem converter o polipeptídeo em fibrilas amilóides. De fato, vários estudos sugerem que intermediários parcialmente enovelados estão envolvidos na fibrilogênese. A apomioglobina (apoMb) de baleia do espermacete é uma proteína bem caracterizada, que forma um intermediário durante o desenovelamento do estado nativo ou após a diluição do estado desenovelado em tampão de enovelamento. A mioglobina é uma proteína altamente solúvel, cujas propriedades do estado nativo não sugerem uma predisposição dessa em formar fibrilas amilóides, corroborado pela organização de sua seqüência de resíduos de aminoácidos em hélices-? bem definidas sem elementos de estrutura em folha-?. Contudo, até a mioglobina forma fibrilas amilóides em certas condições, sugerindo que a capacidade de formar fibrilas seja uma característica comum de toda proteína e, portanto, não estando relacionada a uma estrutura primária específica. Neste projeto, visamos mapear as potenciais interações envolvidas na agregação e formação de fibrilas amilóides em apomioglobinas. Para tanto, apresentamos aqui os estudos dos efeitos de 19 mutantes de apoMb na cinética amiloidogênica da mesma. A indução de fibrilas amilóides foi realizada através da incubação das apoMbs em tampão borato de sódio 50 mM, pH 9 e aquecidas a 65°C. O processo de agregação foi acompanhado por medidas da emissão de fluorescência de Tioflavina T (ThT) e espectroscopia de dicroísmo circular (CD). Outras propriedades morfo-fisicoquímicas das amilóides de apoMb foram também estudadas: energia de ativação da formação de fibrilas, organização estrutural, citotoxicidade, efeito de semeadura, desmontagem por uréia. Nossos resultados mostram que alguns mutantes (7 no total) afetaram a cinética de formação das amilóides, e surpreendentemente, esses efeitos correlacionam-se bem com o efeito que a mutação tem sobre a estabilidade do estado nativo, mas não com o efeito sobre a estabilidade do estado intermediário do enovelamento. As estruturas globais (investigadas por difração de raios-X) das fibrilas formadas pelas ampomioglobinas selvagem e mutantes mostram-se indistinguíveis. Experimentos de citotoxicidade, utilizando um modelo de células neuronais N2A (neuroblastoma de camundongo), e semeadura, confirmam que as diferentes formas de agregados das proteínas são capazes de diminuir a viabilidade celular e de acelerar a formação das fibrilas. Generalizando, nossos resultados suportam a hipótese de que, embora o desenovelamento parcial preceda a formação de fibrilas em apomioglobina, a formação do intermediário de enovelamento não parece ser um passo obrigatório no processo e assim, o enovelamento e a formação de agregados/fibrilas são aparentemente distintos para essa proteína. Abstract: Protein misfolding usually leads to the formation of fibrillar aggregates with extensive ?-sheet structure, commonly termed amyloid fibrils. The hypothesis that the ability to form ordered ?-rich amyloid fibers with identical structures is a generic property of proteins is supported by the fact that even proteins with no connection to disease, such as myoglobin, are able to generate fibrillar structures. Although several proteins are intrinsically disordered, many are properly packed and should unfold totally or partially exposing aggregation-prone regions that can convert the polypeptide into amyloid fibrils. Actually, several studies suggest that partially folded intermediates are involved in fibrillogenesis. Sperm whale apomyoglobin (apoMb) is a well-characterized protein that forms an intermediate after either unfolding from the native state or dilution of the unfolded protein in a folding buffer. Mb is a highly soluble protein whose native state properties do not suggest a predisposition to form amyloid fibrils, corroborated by its amino acid residues sequence organization in well-defined ?-helices with no ?-sheet elements. However, even Mb forms amyloid fibrils under certain conditions, suggesting that the ability to form fibrils is a common feature of all proteins and is not related to a specific primary structure. In this work, we aim to map potential interactions involved in apomioglobin aggregation and fibrils formation. To such aim, we present here the studies of effects on amiloidogenic kinetics of 19 apoMb mutants. The induction of amyloid fibrils was performed by incubating apoMb proteins on 50 mM sodium borate buffer at pH 9 and heat to 65°C. The aggregation process was monitored both by thioflavin T (ThT) emitted fluorescence and circular dichroism (CD) spectroscopy measurements. Other morph-physicochemical properties of apoMb amyloids were also studied: activation energy of fibril formation, structure organization, cytotoxicity, seeding effect, disassembly by urea. Our results show that some mutants (7 in total) affect the amyloid formation kinetics, and surprisingly, these effects are well correlated with the effect that the mutation has on the stability of the native state but not with the effect on the stability of the folding intermediate. The overall structures, probed by X-ray diffraction, of fibers formed by mutants and wild-type apomyoglobin are indistinguishable. Cytotoxicity experiments, using a neuronal cell line model N2A (murine neuroblastoma), and seeding experiments, confirm that different aggregated forms of proteins are capable of decreasing the cell viability and to speed up the formation of fibrils. Generally, our results support the hypothesis that although partially unfolding precedes fibril formation in apomyoglobin, formation of the folding intermediate is not an obligatory step in the process and thus folding and aggregation/fibril formation are apparently distinct in this protein. Doutorado Bioquímica Doutor em Biologia Funcional e Molecular

β-Amyloid (Aβ) is a toxic peptide that has a high propensity for aggregation, and it is often present in the brains of patients with Alzheimer's disease (AD). Bioactive compounds from medicinal plants with neuroprotective properties are of interest in protecting against toxic forms of the Aβ peptide. In the present work, we evaluate the in vitro antiaggregant / disaggregating capacity of amyloid and amyloid-type aggregates by the anthocyanins of Zea mays L. (purple corn). Using the differential pH method, we quantified the total anthocyanin content. With the help of spectrophotometric readings of the amyloid-like protein-Congo Red and Aβ-Congo Red complexes, we evaluated the antiaggregation and disaggregation effects of anthocyanins on amyloid peptides and amyloid derivatives. The results showed a significant activity of antiaggregant / disaggregating capacity by anthocyanins, (33.54 and 26.63%, respectively) which was dependent on the concentration. In addition, the anthocyanin concentrations found of 32.95 and 280.69 mg / L (in aqueous and acidic methanolic medium, respectively), have been compared with anthocyanins and polyphenols from different sources. The results obtained are encouraging to continue evaluating anthocyanins as potential biological agents. El β-amiloide (Aβ) es un péptido tóxico que posee una alta propensión a la agregación, y a menudo se encuentra presente en el cerebro de los pacientes con la enfermedad de Alzheimer (EA). Los compuestos bioactivos de plantas medicinales con propiedades neuroprotectoras, son agentes de especial interés en la protección contra formas tóxicas del péptido Aβ. En el presente trabajo evaluamos la capacidad antiagregante/desagregante de agregados amiloides y derivados amiloides in vitro por parte de las antocianinas de Zea mays L. (maíz morado). Utilizando el método de pH diferencial, cuantificamos el contenido de antocianinas totales. Con ayuda de las lecturas espectrofotométricas de los complejos proteína tipo amiloide- Rojo Congo y Aβ-Rojo Congo, evaluamos los efectos de antiagregación y desagregación por parte de las antocianinas sobre los péptidos amiloide y derivados amiloide. Los resultados obtenidos evidencian una significativa actividad de capacidad antiagregante y desagregante por parte de las antocianinas (33,54 y 26,63 %, respectivamente), la cual es dependiente de concentración. Además, las concentraciones de antocianinas encontradas de 32,95 y 280,69 mg/L (en medio acuoso y metanólico acídico respectivamente), han sido comparadas con antocianinas y polifenoles de distinta fuente. Los resultados obtenidos son alentadores para continuar evaluando a las antocianinas como potenciales agentes biológicos.

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder characterized by initial memory impairments that progress to dementia. In this sense, synaptic dysfunction and loss have been established as the pathological features that best correlate with the typical early cognitive decline in this disease. At the histopathological level, post mortem AD brains typically exhibit intraneuronal neurofibrillary tangles (NFTs) along with the accumulation of amyloid-beta (Abeta) peptides in the form of extracellular deposits. Specifically, the oligomeric soluble forms of Abeta are considered the most synaptotoxic species. In addition, neuritic plaques are Abeta deposits surrounded by activated microglia and astroglia cells together with abnormal swellings of neuronal processes named dystrophic neurites. These periplaque aberrant neurites are mostly presynaptic elements and represent the first pathological indicator of synaptic dysfunction. In terms of losing synaptic proteins, the hippocampus is one of the brain regions most affected in AD patients. In this work, we report an early decline in spatial memory, along with hippocampal synaptic changes, in an amyloidogenic APP/PS1 transgenic model. Quantitative electron microscopy revealed a spatial synaptotoxic pattern around neuritic plaques with significant loss of periplaque synaptic terminals, showing rising synapse loss close to the border, especially in larger plaques. Moreover, dystrophic presynapses were filled with autophagic vesicles in detriment of the presynaptic vesicular density, probably interfering with synaptic function at very early synaptopathological disease stages. Electron immunogold labeling showed that the periphery of amyloid plaques, and the associated dystrophic neurites, was enriched in Abeta oligomers supporting an extracellular location of the synaptotoxins. Finally, the incubation of primary neurons with soluble fractions derived from 6-month-old APP/PS1 hippocampus induced significant loss of synaptic proteins, but not neuronal death. Indeed, this preclinical transgenic model could serve to investigate therapies targeted at initial stages of synaptic dysfunction relevant to the prodromal and early AD., This study was supported by the Instituto de Salud Carlos III (ISCiii) of Spain, co-financed by the FEDER funds from European Union, through grants PI18/01557 (to AG) and PI18/01556 (to JV); by the Junta de Andalucia Consejería de Economía y Conocimiento through grants UMA18-FEDERJA-211 (to AG), P18-RT-2233 (to AG), and US-1262734 (to JV) co-financed by Programa Operativo FEDER 2014–2020; by the Spanish Minister of Science and Innovation grant PID2019-108911RA-100 (to DB-V), Beatriz Galindo program BAGAL18/00052 (to DB-V) grant PID2019-107090RA-I00 (to IM-G), and Ramon y Cajal Program RYC-2017-21879 (to IM-G); and by the Malaga University grants B1-2019_07 (to ES-M) and B1-2019_06 (to IM-G). MM-O held a predoctoral contract from Malaga University and ES-M a postdoctoral contract (DOC_00251) from Junta de Andalucia.

Introducció: Nombrosos estudis suggereixen que el deteriorament cognitiu en el cervell ancià típicament presenta un solapament entre patologies neurodegeneratives i vasculars. La malaltia neurodegenerativa més freqüent seria la malaltia d’Alzheimer (MA) i entre les etiologies vasculars més freqüents es troba l’angiopatia amiloide cerebral (AAC). Les dues entitats comparteixen el dipòsit de beta-amiloide (βA), mentre que en la MA aquest dipòsit es troba en les plaques neurítiques, en l’AAC es troba en les parets vasculars d’artèries i/o capil·lars cerebrals. Justificació ciencítifica: La presència de biomarcadors o l’expressió de proteïnes específiques en el cervell podrien definir o modular l’acumulació de βA en l’AAC i βA i/o tau en la MA. Hipòtesi: Diferents apolipoproteïnes com l’ApoE, la ApoJ i la ApoA-I s’han associat a l’agregació i aclariment cerebral del βA. La primera hipòtesi d’aquesta tesi és que l’expressió i distribució d’aquesta família de proteïnes podrien determinar la localització de l’dipòsit de βA cerebral en plaques parenquimatoses o en parets vasculars corticals o meníngies i capil·lars. D’altra banda, s’han descrit diferents gens com loci de susceptibilitat genètica per a la EA mitjançant estudis d’associació de el genoma complet. Entre ells, el gen CD2AP codifica la proteïna associada a CD2 (CD2AP), de la qual poc es coneix sobre la seva funció en el cervell. La segona hipòtesi de treball és l’existència d’una associació entre l’expressió i distribució de CD2AP, i les característiques neuropatològiques de la MA. Objectius: Conèixer la distribució anatomo-patològica i localització cel·lular dels dipòsits d’aquestes proteïnes en el cervell humà en pacients amb AAC i/o EA. Disseny de l’estudi: Es van realitzar principalment dos estudis immunohistoquímics en cervells humans, un centrat en l’anàlisi de les apolipoproteïnes ApoE, ApoJ i ApoA-I en pacients amb i sense AAC i un altre enfocat en l’estudi de CD2AP en pacients amb i sense EA. Per a això, es van analitzar un total de 101 cervells procedents d’autòpsies realitzades principalment a l’Hospital Universitari Vall d’Hebron, per al segon estudi es van incloure també mostres de la Fundació Centre d’Investigació Malalties Neurològiques (CIEN) de Madrid. Es va analitzar la presència o absència de dipòsit vascular, parenquimatós en forma de plaques o en les cèl·lules (neurones, astròcits, oligodendròcits i cèl·lules endotelials) de les 4 proteïnes. En l’estudi de les apolipoproteïnes, a més es va realitzar una anàlisi del genotip ApoE i, en el de CD2AP, es va realitzar immunofluorescència amb doble tinció CD2AP i Tau-AT8, i Tau-3R en malalts amb Malaltia de Pick. Resultats i conclusions: En el primer estudi, es va identificar la presència de dipòsit vascular i parenquimtós de ApoE i ApoJ, sent el dipòsit de ApoA-I de predomini en capil·lars corticals. La presència d’ApoE en artèries corticals es va associar a l’aparició d’hemorràgia intracranial en pacients amb AAC. L’al·lel APOEε2 es va trobar amb més freqüència en els casos de AAC amb hemorràgia cerebral que en els casos de AAC sense hemorràgia. A més, el genotip APOEε4 estava més representat en pacients amb AAC tipus I (dipòsit βA capil·lar), mentre que els pacients amb AAC tipus II (sense dipòsit βA capil·lar) presentaven APOEε2 amb més freqüència. Pel que fa a el segon estudi, es van identificar dipòsit de CD2AP neuronal en pacients amb MA que es van relacionar amb el dipòsit de proteïna tau fosforilada i estadi neuropatològic de Braak. També es van identificar dipòsits neuronals en pacients amb Malaltia de Pick, mentre que no es van trobar en pacients control o altres taupaties com la degeneració corticobasal i la paràlisi supranuclear progressiva. No es van identificar dipòsits de CD2AP en parets vasculars ni parenquimatosos en forma de plaques en pacients amb dipòsit de βA. Introducción: Numerosos estudios sugieren que el deterioro cognitivo en el cerebro anciano típicamente presenta un solapamiento entre patologías neurodegenerativas y vasculares. La enfermedad neurodegenerativa más frecuente sería la enfermedad de Alzheimer (EA) y entre las etiologías vasculares más frecuentes se encuentra la angiopatía amiloide cerebral (AAC). Ambas entidades comparten el depósito de beta-amiloide (βA), mientras que en la EA este depósito se encuentra en las placas neuríticas en la AAC se encuentra en las paredes vasculares de arterias y/o capilares cerebrales. Justificación ciencítifica: La presencia de biomarcadores o la expresión de proteínas específicas en el cerebro podrían definir o modular la acumulación de βA en la AAC y βA y/o tau en la EA. Hipótesis: Diferentes apolipoproteínas como la ApoE, la ApoJ y la ApoA-I se han asociado a la agregación y aclaramiento cerebral del βA. La primera hipótesis de esta tesis es que la expresión y distribución de esta familia de proteínas podrían determinar la localización del depósito de βA cerebral en placas parenquimatosas o en paredes vasculares corticales o meníngeas y capilares. Por otro lado, se han descrito diferentes genes como loci de susceptibilidad genética para la EA mediante estudios de asociación del genoma completo. Entre ellos, el gen CD2AP codifica la proteína asociada a CD2 (CD2AP), de la que poco se conoce sobre su función en el cerebro. La segunda hipótesis de trabajo es la existencia de una asociación entre la expresión y distribución de CD2AP, y las características neuropatológicas de la EA. Objetivos: Conocer la distribución anatomo-patológica y localización celular de los depósitos de estas proteínas en el cerebro humano en pacientes con AAC y/o EA. Diseño del estudio: Se realizaron principalmente dos estudios inmunohistoquímicos en cerebros humanos, uno centrado en el análisis de las apolipoproteínas ApoE, ApoJ y ApoA-I en pacientes con y sin AAC y otro enfocado en el estudio de CD2AP en pacientes con y sin EA. Para ello, se analizaron un total de 101 cerebros procedentes de autopsias realizadas principalmente en el Hospital Universitari Vall d’Hebron y para el segundo estudio se incluyeron también muestras de la Fundación Centro de Investigación Enfermedades Neurologícas (CIEN) de Madrid. Se analizó la presencia o ausencia de depósito vascular, parenquimatoso en forma de placas o en las células (neuronas, astrocitos, oligodendrocitos y células endoteliales) de las 4 proteínas. En el estudio de apolipoproteínas, además se realizó un anàlisis del genotipo ApoE y, en el de CD2AP, se realizó inmunofluorescencia con doble tinción CD2AP y Tau-AT8, y Tau-3R en enfermos con Enfermedad de Pick. Resultados y conclusiones: En el primer estudio, se identificó la presencia de depósito vascular y parenquimatoso de ApoE y ApoJ, siendo el depósito de ApoA-I de predominio en capilares corticales. La presencia de ApoE en arterias corticales se asoció a la aparición de hemorragia intracraneal en pacientes con AAC. El alelo APOEε2 se encontró con mayor frecuencia en los casos de AAC con HIC que en los casos de AAC sin HIC. Además, el genotipo APOEε4 estaba más representado en pacientes con AAC tipo I (depósito βA capilar), mientras que los pacientes con AAC tipo II (sin depósito βA capilar) presentaban APOEε2 con mayor frecuencia. Respecto al segundo estudio, se identificaron depósito de CD2AP neuronal en pacientes con EA que se relacionaron con el depósito de proteína tau fosforilada y estadio neuropatológico de Braak. También se identificaron depósitos neuronales en pacientes con Enfermedad de Pick, mientras que no se encontraron en pacientes control u otras taupatías como la degeneración corticobasal y la parálisis supranuclear progresiva. No se identificaron depósitos de CD2AP en paredes vasculares ni parenquimatosos en forma de placas en pacientes con depósito de βA. Introduction: Numerous studies suggest that cognitive impairment in the elderly brain typically presents an overlap between neurodegenerative and vascular pathologies. The most common neurodegenerative disease would be Alzheimer’s disease (AD) and among the most common vascular etiologies would be cerebral amyloid angiopathy (CAA). Both entities share the deposit of beta-amyloid (βA),this deposit is found in neuritic plaques in AD while it is found in brain vascular walls in CAA. Scientific reason: The presence of specific proteins biomarkers in the brain could define or modulate the accumulation of βA in CAA and βA and/or tau in AD. Hypothesis: Different apolipoproteins such as ApoE, ApoJ and ApoA-I have been associated with the aggregation and brain clearance of βA. The first hypothesis of this thesis was that the expression and distribution of this family of proteins could determine the location of the deposit of cerebral βA in parenchymal plaques or in cortical or meningeal vascular walls and capillaries. On the other hand, different genes have been described as genetic susceptibility loci for AD by genome-wide association studies. Among them, the CD2AP gene encodes the CD2-associated protein (CD2AP), of which little is known about its function in the brain. The second hypothesis was the existence of an association between the expression and distribution of CD2AP, and the neuropathological characteristics of AD. Objectives: To analyze the anatomo-pathological distribution and cellular location of the deposits of these proteins in the human brain in patients with CAA and/or AD. Study design: Two immunohistochemical studies were mainly carried out in human brains, one focused on the analysis of the apolipoproteins ApoE, ApoJ and ApoA-I in patients with and without AAC and another focused on the study of CD2AP in patients with and without AD. A total of 101 brains from autopsies performed mainly at the Vall d’Hebron University Hospital were analyzed and samples from the Madrid Neurological Diseases Research Center Foundation (CIEN) in Madrid were also included for the second study. The presence or absence of vascular, parenchymal deposit in the form of plaques or in the cells (neurons, astrocytes, oligodendrocytes and endothelial cells) of the 4 proteins was analyzed. In the apolipoprotein study, an analysis of the ApoE genotype was also carried out. In the CD2AP study, Tau-3 R in patients with Pick’s disease and CD2AP and Tau-AT8 immunofluorescence studies were added in CD2AP study. Results and conclusions: In the first study, the presence of vascular and parenchymal deposit of ApoE and ApoJ was identified, being the deposit of ApoA-I predominantly in cortical capillaries. The presence of ApoE in cortical arteries was associated with the appearance of intracranial hemorrhage in patients with CAA. The APOEε2 allele was found more frequently in CAA cases with hemorrhage than in CAA cases without hemorrhage. Furthermore, the APOEε4 genotype was more represented in CAA type I patients, while patients with CAA type II cases presented more frequently APOEε2. Regarding the second study, neuronal CD2AP deposition was identified in AD patients that were related to phosphorylated tau protein deposition and Braak’s neuropathological stage. Neural deposits were also identified in patients with Pick’s disease, while they were not found in control patients or other taupathies such as corticobasal degeneration and progressive supranuclear palsy. No CD2AP deposits on vascular walls or plaques in the parenchyma were identified in patients with βA deposition. Universitat Autònoma de Barcelona. Programa de Doctorat en Cirurgia i Ciències Morfològiques

RESUMEN El β-amiloide (Aβ) es un péptido tóxico que posee una alta propensión a la agregación, y a menudo se encuentra presente en el cerebro de los pacientes con la enfermedad de Alzheimer (EA). Los compuestos bioactivos de plantas medicinales con propiedades neuroprotectoras, son agentes de especial interés en la protección contra formas tóxicas del péptido Aβ. En el presente trabajo evaluamos la capacidad antiagregante/desagregante de agregados amiloides y derivados amiloides in vitro por parte de las antocianinas de Zea mays L. (maíz morado). Utilizando el método de pH diferencial, cuantificamos el contenido de antocianinas totales. Con ayuda de las lecturas espectrofotométricas de los complejos proteína tipo amiloide- Rojo Congo y Aβ-Rojo Congo, evaluamos los efectos de antiagregación y desagregación por parte de las antocianinas sobre los péptidos amiloide y derivados amiloide. Los resultados obtenidos evidencian una significativa actividad de capacidad antiagregante y desagregante por parte de las antocianinas (33,54 y 26,63 %, respectivamente), la cual es dependiente de concentración. Además, las concentraciones de antocianinas encontradas de 32,95 y 280,69 mg/L (en medio acuoso y metanólico acídico respectivamente), han sido comparadas con antocianinas y polifenoles de distinta fuente. Los resultados obtenidos son alentadores para continuar evaluando a las antocianinas como potenciales agentes biológicos. ABSTRACT β-Amyloid (Aβ) is a toxic peptide that has a high propensity for aggregation, and it is often present in the brains of patients with Alzheimer's disease (AD). Bioactive compounds from medicinal plants with neuroprotective properties are of interest in protecting against toxic forms of the Aβ peptide. In the present work, we evaluate the in vitro antiaggregant / disaggregating capacity of amyloid and amyloid-type aggregates by the anthocyanins of Zea mays L. (purple corn). Using the differential pH method, we quantified the total anthocyanin content. With the help of spectrophotometric readings of the amyloid-like protein-Congo Red and Aβ-Congo Red complexes, we evaluated the antiaggregation and disaggregation effects of anthocyanins on amyloid peptides and amyloid derivatives. The results showed a significant activity of antiaggregant / disaggregating capacity by anthocyanins, (33.54 and 26.63%, respectively) which was dependent on the concentration. In addition, the anthocyanin concentrations found of 32.95 and 280.69 mg / L (in aqueous and acidic methanolic medium, respectively), have been compared with anthocyanins and polyphenols from different sources. The results obtained are encouraging to continue evaluating anthocyanins as potential biological agents.

In Alzheimer’s disease (AD), and other tauopathies, microtubule destabilization compromises axonal and synaptic integrity contributing to neurodegeneration. These diseases are characterized by the intracellular accumulation of hyperphosphorylated tau leading to neurofibrillary pathology. AD brains also accumulate amyloid-beta (Aβ) deposits. However, the effect of microtubule stabilizing agents on Aβ pathology has not been assessed so far. Here we have evaluated the impact of the brain-penetrant microtubule-stabilizing agent Epothilone D (EpoD) in an amyloidogenic model of AD. Three-month-old APP/PS1 mice, before the pathology onset, were weekly injected with EpoD for 3 months. Treated mice showed significant decrease in the phospho-tau levels and, more interesting, in the intracellular and extracellular hippocampal Aβ accumulation, including the soluble oligomeric forms. Moreover, a significant cognitive improvement and amelioration of the synaptic and neuritic pathology was found. Remarkably, EpoD exerted a neuroprotective effect on SOM-interneurons, a highly AD-vulnerable GABAergic subpopulation. Therefore, our results suggested that EpoD improved microtubule dynamics and axonal transport in an AD-like context, reducing tau and Aβ levels and promoting neuronal and cognitive protection. These results underline the existence of a crosstalk between cytoskeleton pathology and the two major AD protein lesions. Therefore, microtubule stabilizers could be considered therapeutic agents to slow the progression of both tau and Aβ pathology., This study was supported by Instituto de Salud Carlos III (ISCiii) of Spain, co-financed by FEDER funds from European Union, through grants PI15/00796 and PI18/01557 (both to AG), PI15/00957 and PI18/01556 (both to JV), and CIBERNED (CB06/05/1116 to AG and CB06/05/0094 to JV); by Malaga University grant PPIT.UMA.B1.2017/26 (to RSV). JJFV and CMC were supported by FPU PhD fellowships (Spanish Ministry of Science, Innovation and Universities). RSV held a postdoctoral contract from Malaga University. IMG is recipient of a senior postdoctoral contract from Ramon y Cajal Program (Spain Government).

Mitochondria contribute to shape intraneuronal Ca2+ signals. Excessive Ca2+ taken up by mitochondria could lead to cell death. Amyloid beta (A beta) causes cytosolic Ca2+ overload, but the effects of A beta on mitochondrial Ca2+ levels in Alzheimer's disease (AD) remain unclear. Using a ratiometric Ca2+ indicator targeted to neuronal mitochondria and intravital multiphoton microscopy, we find increased mitochondrial Ca2+ levels associated with plaque deposition and neuronal death in a transgenic mouse model of cerebral beta -amyloidosis. Naturally secreted soluble A beta applied onto the healthy brain increases Ca2+ concentration in mitochondria, which is prevented by blockage of the mitochondrial calcium uniporter. RNA-sequencing from post-mortem AD human brains shows downregulation in the expression of mitochondrial influx Ca2+ transporter genes, but upregulation in the genes related to mitochondrial Ca2+ efflux pathways, suggesting a counteracting effect to avoid Ca2+ overload. We propose lowering neuronal mitochondrial Ca2+ by inhibiting the mitochondrial Ca2+ uniporter as a novel potential therapeutic target against AD. Calvo-Rodriguez et al. show elevated calcium levels in neuronal mitochondria in a mouse model of cerebral beta -amyloidosis after plaque deposition, which precede rare neuron death events in this model. The mechanism involves toxic extracellular A beta oligomers and the mitochondrial calcium uniporter.

La enfermedad de Alzheimer (EA) es la proteinopatía neurodegenerativa más prevalente y para la que no existe ningún tratamiento. Se caracteriza por la acumulación cerebral de péptido beta-amiloide (Aβ) en forma de placas extracelulares, y de tau hiperfosforilado (fosfo-tau) como ovillos neurofibrilares intraneuronales. Junto con pérdida sináptica y neuronal además hay un proceso neuroinflamatorio mediado por activación de células gliales. Aún no se conoce bien el papel de la microglía y astroglía en la patogénesis de la EA, especialmente frente a la patología tau, por lo que descifrar la naturaleza molecular y el efecto de distintas cepas tóxicas de tau supondría una nueva oportunidad terapéutica, sobre todo considerando el fracaso de las terapias centradas en Aβ. El objetivo de esta Tesis Doctoral es analizar a nivel celular, subcelular y molecular, la progresión de la patología de tau y su efecto sobre la microglía y la astroglía, así como la pérdida sináptica y neuronal en el hipocampo de modelos de tauopatía (P301S y ThyTau22). Además, se ha estudiado el efecto de un estabilizador de microtúbulos, epotilona D, en un modelo amiloidogénico (APP/PS1) para dilucidar la posible interacción entre las patologías Aβ y tau. Este trabajo demuestra que las especies de fosfo-tau del modelo P301S desencadenan una respuesta glial más agresiva y un daño neuronal (especialmente mitocondrial) más severo que las de ThyTau22. La microglía activada de P301S adquiere un fenotipo DAM mientras que en ThyTau22 existe escasa reactividad microglial con un perfil degenerativo. Finalmente, la estabilización microtubular en el modelo APP/PS1 protege del deterioro de la función cognitiva, disminuyendo la patología neurítica (axonal y sináptica) y la pérdida neuronal, reduciendo la acumulación de fosfo-tau y de Aβ. Por tanto, los estabilizadores de microtúbulos pueden considerarse potenciales candidatos terapéuticos para la EA al actuar en puntos de interacción entre Aβ y tau.