A Doença de Alzheimer (DA) é uma doença cerebral progressiva que resulta em prejuízos na memória e disfunção cognitiva global. Entre as principais características neuropatológicas associadas a DA estão à presença de placas senis, emaranhados neurofibrilares e a hiperfosforilação da proteína Tau. A hiperativação do sistema glutamatérgico tem sido implicada na fisiopatologia da DA. O excesso de glutamato na fenda sináptica causa hiperativação do seu receptor ionótropico N-metill-D-aspartato (NMDA) o que favorece o aumento do influxo de cálcio e morte neuronal. A administração intracerebral de ácido ocadáico (AO) causa alterações morfológicas e funcionais similares à DA. O AO promove a inibição da proteína fosfatase 2A (PP2A) favorecendo as atividades cinásicas de proteínas como a cinase dependente de ciclina 5 (Cdk5). A memantina (MN) é uma das principais drogas utilizadas no tratamento da DA e o seu mecanismo de ação envolve um antagonismo não competitivo de baixa afinidade pela subunidade NR2B do receptor NMDA. Neste trabalho, foram avaliados efeitos do pré-tratamento com MN em um modelo semelhante a DA induzido pela administração intrahipocampal de AO em ratos. O pré-tratamento com MN preveniu o déficit na memória especial causado pela infusão intrahipocampal de AO. Os mecanismos envolvidos nestes efeitos neuroprotetores envolvem a prevenção do aumento de glutamato no liquido cefalorraquidiano, juntamente com a regulação da expressão de Cdk5 e em conseqüência a prevenção do aumento da fosforilação de Tau. Desta maneira, a MN pode ser um alvo terapêutico para prevenir as alterações comportamentais e neuroquímicas em um modelo similar a DA induzido pelo AO. / Alzheimer's disease (AD) is a progressive brain disease that causes memory loss and global cognitive dysfunction. The neuropathological alterations associated with AD include senile plaques, neurofibrillary tangles and Tau protein hyperphosphorylation. The glutamatergic system is implicated in the pathophysiology of AD. Indeed, the excessive glutamate levels in the synaptic cleft may cause hyperactivation of glutamate ionotropic N-metill-Daspartate (NMDA), which favors increase calcium influx and neuronal death. The intracerebral administration of okadaic acid (OA) causes morphological and functional alterations similar to AD. The OA inhibits the protein phosphatase 2A (PP2A) thus overstimulating the kinases activities. Memantine (MN) is a drug currently used in the treatment of AD, which mechanism involves a noncompetitive low affinity antagonism for NR2B subunit of NMDA receptors. In this work we evaluate the effects of pretreatment with MN in an AD-like model in rats induced by intrahippocampal administration of OA. The pretreatment with MN could prevent the spatial memory deficits caused by OA intrahipocampal administration in rats. The mechanisms underlying this neuroprotective effects involves the prevention of the increase in brain glutamate levels along with regulation of Cdk5 and, in consequence, downstream phosphorylation of Tau (ser199/202) protein. To conclude, MN has potential therapeutic role in preventing behavioral and neurochemical alterations caused by an AD like model induced by OA.

As doenças neurodegenerativas (DN) estão entre as principais causas de mortalidade e morbidade nos países ocidentais. Não há ainda um tratamento definitivo para estas neuropatias, mas os estudos têm indicado mecanismos comuns de toxicidade que incluem disfunção mitocondrial, estresse oxidativo e apoptose. Assim, as mitocôndrias constituem alvos importantes para futuras estratégias de neuroproteção a fim de tratar, prevenir ou até mesmo retardar a neurodegeneração. Neste contexto, o canabidiol (CBD), um constituinte não psicoativo da Cannabis sativa e cuja propriedade neuroprotetora tem sido sugerida por diferentes estudos, surge como uma alternativa bastante promissora. Diferentes mecanismos moleculares podem estar envolvidos na neuroproteção exercida pelo CBD. Embora o potencial efeito benéfico do canabidiol com relação às doenças neurodegenerativas já tenha sido sugerido, não há ainda estudos que abordem precisamente os mecanismos de proteção contra a toxicidade mitocondrial cerebral, evento chave no processo neurodegenerativo. O presente estudo teve como objetivo investigar os efeitos do CBD em mitocôndrias cerebrais de rato, bem como possíveis mecanismos de neuroproteção. Foram avaliados os seguintes parâmetros: função mitocondrial, estresse oxidativo mitocondrial e transição de permeabilidade de membrana mitocondrial (TPMM). Os resultados obtidos sugerem que o canabidiol é capaz de proteger as mitocôndrias cerebrais contra o intumescimento osmótico induzido por cálcio/fosfato, contra a produção de H2O2 induzida por terc-butil hidroperóxido e contra a peroxidação lipídica induzida por Fe2+ e citrato. A captação mitocondrial de cálcio e a capacidade fosforilativa não foram afetadas. / Neurodegenerative diseases (ND) are among the leading causes of mortality and morbidity in Western countries. There is not a definitive treatment for these neuropathies, but studies have indicated mechanisms of toxicity which include mitochondrial dysfunction, oxidative stress and apoptosis. Therefore, mitochondria are important targets for future neuroprotective strategies to treat, prevent or even slow the neurodegeneration. In this context, cannabidiol (CBD), a constituent of non-psychoactive Cannabis sativa and whose neuroprotective property has been suggested by different studies, emerges as a promising alternative. Different molecular mechanisms may be involved in the neuroprotection exerted by CBD. Although the potential beneficial effects of cannabidiol in relation to neurodegenerative diseases has already been suggested, there are no studies addressing specifically the mechanisms of protection against mitochondrial toxicity brain, a key event in the neurodegenerative process. This study aimed to investigate the effects of CBD on rat brain mitochondria, as well as the mechanisms of neuroprotection. The following parameters were evaluated: mitochondrial function, mitochondrial oxidative stress and permeability transition of the mitochondrial membrane (MPT). The results suggest that cannabidiol can protect brain mitochondria against: the osmotic swelling induced by calcium/phosphate, the production of H2O2 induced by tert-butyl hydroperoxide and the lipid peroxidation induced by Fe2+ and citrate. The mitochondrial calcium uptake and phosphorylative capacity were not affected.

Submitted by Neusa Fagundes (neusa.fagundes@unioeste.br) on 2018-03-06T19:51:35Z No. of bitstreams: 2 Suélyn_Koerich2017.pdf: 1381882 bytes, checksum: 2404add8c5fc58aba0ebaa970b5c23be (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-03-06T19:51:36Z (GMT). No. of bitstreams: 2 Suélyn_Koerich2017.pdf: 1381882 bytes, checksum: 2404add8c5fc58aba0ebaa970b5c23be (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-12-15 / Fundação Araucária / Tamoxifen is classified as selective estrogen receptor modulador (SERM), being used in the treatment and prevention of breast cancer. Although presents many side effects, tamoxifen is a therapy of choice for the treatment of breast cancer in the postmenopausal period. The quercetin, a flavonoid with antioxidant potent effect, has benefits on the adverse effects caused by tamoxifen, especially those related to lipoperoxidation. The study of the neuroprotective effect of quercetin on brain bioenergetics is important for the recognition of the mechanisms involved. Thus, the objective of this work was to analyze the tamoxifen effects on glucose metabolism in the brain and the influence of quercetin coadministered with tamoxifen on passage of glucose from the cerebrospinal fluid to the brain of ovariectomized rats submitted to tamoxifen treatment. For this, bilaterally ovariectomized rats were separated into groups and treated with canola oil (1.0 mL.Kg-1) tamoxifen (5 mg.Kg-1), quercetin (22.5 mg.Kg-1) and the coadministration of tamoxifen (5 mg.Kg-1) plus quercetin (22.5 mg.Kg-1), orally for 14 days. The blood, cerebrospinal fluid, cerebellum, cortex, and hippocampus were collected for glucose lactate and pyruvate dosage as well copper and ceruloplasmin. It was observed that tamoxifen increased blood glucose levels and that co-administration with quercetin brought these levels close to the values of the control group. In cerebrospinal fluid, the quercetin administration didn´t influence glucose levels and in the different brain regions, both quercetin alone such as coadministration with tamoxifen significantly decreased glucose values when compared to the group that received only tamoxifen. The coadministration of quercetin with tamoxifen was shown be effective not only in maintenance glycemic levels but also in cerebral glucose levels. / O tamoxifeno é classificado como modulador seletivo de receptor de estrógeno (SERM), sendo utilizado no tratamento e na prevenção do câncer de mama. Embora apresente muito efeitos colaterais, o tamoxifeno é a terapia de escolha para o tratamento do câncer de mama no período da pós-menopausa. A quercetina, um flavonóide com potente efeito antioxidante, traz benefícios diante dos efeitos adversos gerados pelo tamoxifeno, principalmente aqueles relacionados à lipoperoxidação. O estudo do efeito neuroprotetor da quercetina sobre a bioenergética cerebral é importante para o reconhecimento dos mecanismos envolvidos. Assim, o objetivo deste trabalho foi analisar o efeito do tamoxifeno sobre o metabolismo da glicose no cérebro e a influência da quercetina coadministrada com o tamoxifeno na passagem da glicose do líquido cefalorraquidiano para o cérebro de ratas ovariectomizadas submetidas ao tratamento com tamoxifeno. Para isso, ratas ovariectomizadas bilateralmente foram separadas em grupos e tratadas com óleo de canola (1,0 mL. Kg-1), (tamoxifeno (5 mg.kg-1), quercetina (22,5 mg.kg-1) e a coadministração de tamoxifeno (5 mg.kg-1) mais quercetina (22,5 mg.kg-1) por via oral, durante 14 dias. O sangue, o líquido cefalorraquidiano, o cerebelo, o córtex e o hipocampo foram coletados para a dosagem de glicose, lactato e piruvato, bem como cobre e ceruloplasmina. Foi observado que o tamoxifeno aumentou os níveis de glicose no sangue e que a coadministração com quercetina trouxe esses níveis próximos aos valores do grupo controle. No líquido cefalorraquidiano, a administração de quercetina não influenciou nos níveis de glicose e nas diferentes regiões do cérebro, tanto a quercetina isoladamente, como a coadministração da mesma com o tamoxifeno, diminuiu significativamente os valores de glicose, quando comparados ao grupo que recebeu apenas tamoxifeno. A coadministração de quercetina com o tamoxifeno mostrou-se eficaz não só na manutenção dos níveis glicêmicos, como também nos níveis de glicose cerebral.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-04-20T12:04:36Z No. of bitstreams: 1 williantassiogomesnovato.pdf: 21495870 bytes, checksum: 315183cde6b8aeeb62723ff350f4bc9e (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-20T13:10:32Z (GMT) No. of bitstreams: 1 williantassiogomesnovato.pdf: 21495870 bytes, checksum: 315183cde6b8aeeb62723ff350f4bc9e (MD5) / Made available in DSpace on 2017-04-20T13:10:32Z (GMT). No. of bitstreams: 1 williantassiogomesnovato.pdf: 21495870 bytes, checksum: 315183cde6b8aeeb62723ff350f4bc9e (MD5) Previous issue date: 2017-01-27 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A presente tese propõe um estudo de alvos específicos recorrentes a quimioterapia da Doença de Alzheimer. Estudos apontam que complexos de platina(II) podem ser utilizados como compostos anti-aglomeração de peptídeos β-amilóide que se compactam entre as fendas sinápticas, impedindo a efetivação do estímulo do potencial de ação, e com isso, inativação da região cerebral. Estas espécies são portadoras de sítios com potencial probabilidade de coordenação metálica, onde a ordem de reatividade de cada aminoácido foi investigada. Para tanto, foram elaborados experimentos para validação de resultados de barreira de energia livre de ativação utilizando Teoria do Funcional de Densidade (DFT) para a hidrólise do complexo pt01, Pt(ofen)Cl2 ( ΔG# 1,EXP=26,94; ΔG#1,TEO=25,54 kcal mol-1) e também para substituição nucleofílica de segunda ordem de cloreto por histidinas (HIS) ( ΔG# 1,EXP=20,73; G# 1,TEO=20,74 kcal mol-1). Após ajuste metodológico, o mesmo protocolo foi utilizado para comparar a reatividade para todos os aminoácidos (AA) que constituem a espécie β-amilóide dada por MET > LYS > HIS > GLU > ASP, e termodinamicamente é pronunciado com a comparação LYS > HIS > GLU > ASP > MET. Para verificar o perfil estrutural para agregados peptídicos e consequentemente, atribuição de um valor energético em cada evento recorrente entre os ligantes derivados de fenantrolina, batofenantrolina, curcumina e pt01, a metodologia de Docking Molecular e cálculos de Dinâmica Molecular (DM) seguidos de cálculos MM-PBSA foram necessários, dos quais foram indicativos de desestruturação da agregação inicial. Após análise estrutural dada por resultados de DM, foi possível observar a alta flexibilidade dos resíduos na região central do peptídeo quando é inserido o ligante curcumina, na forma enoláto (CUR-1). O modelo de simulação mais destacável foi 9PEP@pt01, onde é observado o completo colapso do agregado, obtendo valor mais negativo de GGBTOT = -39±7 kcal mol-1, valor este que porta 19 kcal mol-1 mais negativo comparado aos outros ligantes, e portanto, evidenciando o papel da platina como potencial fator utilizado em estágios de aglomeração inicial, mesmo sem a coordenação metálica com os sítios de MET, LYS ou HIS. / The present thesis proposes a study of specific targets recurrent to Alzheimer's disease chemotherapy. Studies show that platinum (II) complexes can be used as anti-agglomeration compounds of -amyloid peptides that are compacted between the synaptic clefts, preventing the activation of the action potential stimulus, and thus, inactivation of the brain region. These species are carriers of sites with potential probability of metallic coordination, where the order of reactivity of each amino acid was investigated. For this, experiments were developed to validate the results of activation free energy barrier using Density Functional Theory (DFT) for the hydrolysis of the pt01 complex, Pt(ofen)Cl2 ( ΔG# 1,EXP=26,94; ΔG# 1,TEO=25,54 kcal mol-1) and also for second order nucleophilic substitution of histidines (HYS) (ΔG# 1,EXP=20,73; ΔG# 1,THEO=20,74 kcal mol-1). After methodological adjustment, the protocol was used to compare the reactivity for all aminoacids (AA) which constitute the -amyloid species given by MET > LYS > HIS > GLU > ASP, and is thermodynamically pronounced with the comparison LYS > HIS > GLU > ASP > MET. To verify the structural profile for peptide aggregates and, consequently, assign an energetic value in each recurrent event among the ligands derived from phenanthroline, batofenanthroline, curcumin and pt01, the Molecular Docking methodology and Molecular Dynamics (DM) calculations followed by MM-PBSA approximation with indicative of the destructuring of the initial aggregation. After structural analysis given by DM results, it was possible to observe the high flexibility of the residues in the central region of the peptide when the ligand curcumin is inserted, in the enolate form (CUR-1). The most detachable simulation model was 9PEP @ pt01, where the complete collapse of the aggregate is observed, obtaining a more negative value of GGBTOT = -39±7 kcal mol-1. This value carries 19 kcal mol-1 plus negative compared to other ligands, and therefore, evidencing the role of platinum as a potential factor used in initial agglomeration stages, even without metallic coordination with the MET, LYS and HYS sites.

Ces travaux de recherche s’inscrivent dans le cadre du développement de nouvelles surfaces biocompatibles capables de contrôler l’adhésion d’agents pathogènes responsables de maladies neurodégénératives telles que les maladies de Creutzfeld Jacob, Alzheimer, Parkinson et Lewis. Deux axes de recherche ont été privilégiés. Notre approche se focalise en amont des dosages sur l’amélioration des procédures de stockage des prélèvements biologiques réalisés dans des tubes de type Eppendorf. Ces tubes en polypropylène induisent une perte du matériel génétique de plus de 70% accentuant la faible concentration en agent pathogène pour la détection immunoenzymatique. Dans le but de réduire les phénomènes indésirables d’adhésion des agents pathogènes à la surface des supports de stockage, deux voies de traitement ont été envisagées dans ce travail de thèse. La première consiste à modifier la surface du tube Eppendorf en une étape par décharge plasma fluoré, la seconde à créer de nouvelles surfaces hydrophiles en deux étapes couplant la technique des plasmas froids au greffage de polymères, les agents pathogènes pouvant être hydrophiles ou hydrophobes. Avec cette dernière technique, une voie originale a été abordée de part l’utilisation de solutions de greffage complexes composées à la fois de polymères et de molécules tensioactives. Les surfaces ainsi obtenues présentent une nano-structuration. Toutes les étapes de modification de la surface interne des tubes de stockage ont été caractérisées. Ces surfaces sont alors décrites selon leur caractère hydrophile ou hydrophobe grâce à la détermination des énergies de surface polaire et apolaire, selon leur charge de surface obtenue par mesure du potentiel d’écoulement, selon leur composition chimique déterminée par spectroscopie à photoélectrons X (XPS) et enfin selon leur topographie et leur rugosité relevées par microscopie à force atomique (AFM). Les interactions entre les groupements fonctionnels ainsi obtenus à la surface des tubes de stockage après les divers traitements et les protéines antigéniques considérées ont été interprétées en se référant aux différents modèles de l’adhésion pour des gammes de pH proches des protocoles biologiques usuels. Afin de s’assurer que ces nouvelles surfaces permettent bien une diminution de l’adhésion des agents infectieux sur la paroi interne des tubes de polypropylène, des analyses immunoenzymatiques ont été réalisées au sein des centres hospitaliers participant au projet STREP NEUROSCREEN n° LSHB-CT 2006-03 7719 (CRPP de Liège et CHU de Lyon). Ces analyses ont permis de montrer que la modification des surfaces entraîne une diminution de l’absorption des agents pathogènes jusqu'à 100% permettant ainsi une meilleure détection. / The research work presented in this thesis considers the development of newµbiocompatible surfaces that are able to control the adhesion of specific proteins responsible for the development of neurodegenerative diseases such as Creutzfeldt–Jakob, Alzheimer, Parkinson and Lewis body disease. Our approach was focused on problems prior to the detection step, which were never considered before, particularly on the improvement of Eppendorf tubes that are used for the storage of body fluids like cerebrospinal fluid and blood. Namely these tubes made of polypropylene induce the depletion of biological material, in some cases even over 70%, resulting in a low concentration of these proteins for the further immunoenzymatic detection. With the purpose to reduce the adhesion of specific proteins on the surface of supports, two courses of treatments were anticipated. The first one consists of surface modification by highly reactive fluorine plasma treatment and the second one incorporates development of new hydrophilic surfaces by coupling two techniques, plasma activation and subsequent grafting of polymer materials. With the latter approach, an original way of surface modification has been attained by using complex solutions of polymers and surfactants that permits controlled configuration of nanostructured surfaces. All steps of surface modifications were well characterized by different physicochemical methods. The surface hydrophilic/hydrophobic character was determined by measurements of polar and apolar surface energy, surface charge by magnitude of zeta potential, surface chemistry was evaluated by x-ray photoelectron spectroscopy (XPS), while the surface roughness and topography were monitored by atomic force microscopy (AFM). The interactions between functional groups of treated supports and proteins were interpreted referring to different models of adhesion established for a range of pH values close to the classical biological protocols. Finally, in order to validate that the new surfaces are able to prevent or decrease the adhesion of neurodegenerative agents on the surfaces of Eppendorf tubes, the immunoenzymatic analyses were carried out in hospital centres of partners that were participating to the project STREP NEUROSREEN n° LSHB-CT-2006-03 7719 (Centre de Recherche sur les Protéines Prion; Liege (ULG), Hospices Civils de Lyon (CHUL) and Lancaster University (L-UNI)). These analyses showed that the treatments led to a decrease of antigen adsorption up to 100%, enabling (allowing) better detection of pathogenic agents.

Les maladies neurodégénaratives (MNDs) sont les troubles neurologiques les plus fréquents chez l’homme et touchent des millions de personnes à travers le monde. Elles affectent le fonctionnement du système nerveux de façon chronique et progressive et conduisent souvent au décès du malade. L’évolution de ces maladies est très variable et les traitements disponibles actuellement ne permettent pas de modifier leur progression mais seulement d’atténuer les manifestations symptomatiques.Les récepteurs σ1 correspondent à une classe unique de récepteurs transmembranaires du réticulum endoplasmique. Exprimés au niveau du SNC et en particulier dans les neurones, les lymphocytes et les oligodendrocytes, ces récepteurs sont connus pour être impliqués dans la régulation de nombreux neurotransmetteurs. Même si les mécanismes de transduction après leur activation ne sont pas complètement élucidés, de plus en plus d’études mettent en évidence le potentiel thérapeutique de ces récepteurs. En effet, depuis leur découverte, les récepteurs σ1 ont été impliqués dans de nombreuses pathologies dont des MNDs.Ces travaux de thèse s’inscrivent donc dans ce contexte. Le projet consistait à concevoir, synthétiser et évaluer de nouveaux ligands σ1 pour une utilisation en neuroprotection. Les dérivés tétrahydroquinolin-hydantoïnes préalablement conçus au laboratoire, ont montré des affinités nanomolaires envers la protéine σ1, une bonne sélectivité, une faible cytotoxicité et des propriétés ADME compatibles avec un développement thérapeutique. Evalués dans différents modèles, ces composés ont montrés des propriétés anti-inflammatoires associées à une action neuroprotective. Cependant, bien que les Tic-hydantoïnes soient des composés chimiquement stables, ils ont montré une faible stabilité métabolique. Trois nouvelles familles de composés ont donc été conçues et synthétisées afin de pallier à ces problèmes. Leurs affinités, sélectivités, cytotoxicités et propriétés ADME ont été évaluées. Des tests comportementaux ont également été réalisés sur les composés les plus intéressants afin de déterminer leur profil agoniste ou antagoniste. Finalement, le meilleur candidat, évalué dans un modèle in vivo de sclérose en plaques, a montré des propriétés neuroprotectrices intéréssantes. / Neurodegenerative diseases are the most common neurological disorders in humans, affecting millions of people worldwide. They affect the nervous systems in chronic and progressive way and often lead to the death of the patient. The evolution of these diseases is highly variable and currently available treatments do not alter their growth but only moderate symptomatic manifestations.Sigma-1 receptors represent a structurally unique class of transmembrane receptors of the endoplasmic reticulum. Expressed in the central nervous system and especially in neurons, lymphocytes and oligodendrocytes, these receptors are known to be involved in the regulation of numerous neurotransmitters. Even if the signal transduction pathway after activation of σ1 receptors is not completely understood, more and more evidences suggest that they represent a potential therapeutic target in many diseases. Indeed, since their discovery, the σ1 receptors have been implicated in various pathologies including neurodegenerative disorders. Thus, it is in this context that our interest is focused on the conception and synthesis of novel σ1 receptors ligands for the treatment of neurodegenerative diseases. Fused and optimized tetrahydroquinoline-hydantoin derivatives designed in our laboratory showed nanomolar σ1 affinity, σ2 /σ1 selectivity, very low cytotoxicity and ADME properties compatible with therapeutic development. Evaluated in different models, these compounds showed an anti-inflammatory activity associated with a neuroprotective action. However, while the Tic-Hydantoin derivatives are chemically stable, they showed a low metabolic stability. Thus, three novel families of compounds were synthesized in order to compensate for these problems. Their affinities, selectivities, cytotoxicities and their ADME properties were evaluated. Behavioural testing was carried out on the most interesting compounds to determine the agonist or antagonist profil. Finally, evaluated in in vivo model of multiple sclerosis, the best compound showed interesting neuroprotective properties.

Les protéoformes désignent toutes les formes sous lesquelles une protéine peut être présente. Cela inclut les formes portant des modifications post-traductionnelles, les isoformes et les formes résultant d’épissages alternatifs. Ces modifications peuvent influer sur la fonction d’une protéine, d’où l’intérêt de développer des méthodes sensibles, spécifiques et robustes de quantification de protéoformes pour une meilleure compréhension de mécanismes pathologiques ou la recherche de biomarqueurs. L’objectif de ce travail a été d’exploiter les avantages de la spectrométrie de masse à haute résolution pour la caractérisation et la quantification de protéoformes de protéines associées à des maladies neurodégénératives. Nous nous sommes principalement intéressés à deux protéines, la C9ORF72 et l’alpha-synucléine. Dans un premier temps, une méthode de quantification des deux isoformes de la C9ORF72 a été développée et appliquée à une cohorte de 43 cerveaux humains comprenant des cas de démences fronto-temporale (DFT) avec ou sans mutation C9ORF72. Les résultats obtenus montrent pour la première fois par spectrométrie de masse une diminution d’environ 50% de l’isoforme longue de la C9ORF72 en présence de la mutation. Dans une seconde étape, nous avons élargi l’analyse à la quantification multiplexe de 49 protéines cérébrales potentiellement impliquées dans les DFT. En parallèle, nous nous sommes intéressés aux formes tronquées de l’alpha-synucléine. Leur quantification a été réalisée par une approche top-down dans des tissus cérébraux et une approche par bottom-up dans le liquide céphalorachidien (LCR). Enfin, l’analyse a été étendue à la quantification multiplexe de l’alpha-synucléine et de la protéine tau du LCR. / Proteoforms describe the complexity of protein forms. This includes forms with post-translational modifications, isoforms and forms resulting from alternative splicing. These modifications can influence the function of a protein, hence the interest in developing sensitive, specific and robust methods for the quantification of proteoforms for a better understanding of pathological mechanisms or biomarkers discovery. The objective of this work was to take advantage of high-resolution mass spectrometry for the characterization and quantification of proteoforms associated with neurodegenerative diseases. We mainly focused on two proteins, C9ORF72 and alpha-synuclein. First, a method for quantifying the two C9ORF72 isoforms was developed and applied to a cohort of 43 human brains including cases of frontotemporal dementia (FTD) with or without C9ORF72 mutation. The results obtained show for the first time by mass spectrometry a decrease of about 50% of the long isoform of C9ORF72 in the presence of the mutation. Then, we extended the analysis to the multiplex quantification of 49 brain proteins potentially involved in FTD. In parallel, we focused on the truncated forms of alpha-synuclein. Their quantification was performed by a top-down approach in brain tissue and a bottom-up approach in cerebrospinal fluid (CSF). Finally, the analysis was extended to the multiplex quantification of alpha-synuclein and tau protein in CSF.

Microsatellites, or simple tandem repeat sequences, occur naturally in the human genome and have important roles in genome evolution and function. However, the expansion of microsatellites is associated with over two dozen neurological diseases. A common denominator among the majority of these disorders is the expression of expanded tandem repeat-containing RNA, referred to as xtrRNA, which can mediate molecular disease pathology in multiple ways. Frontotemporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) are two fatal neurodegenerative diseases with significant clinical, neurological and genetic overlap thus referred to as C9FTD/ALS. Currently, gaps in the study of the underlying disease mechanisms persist, which can aid in the identification of promising therapeutic approaches. Access to simple models of neurological repeat expansion disease is critical for investigating biochemical mechanisms and for early therapeutic discovery. To better understand the molecular pathology of C9FTD/ALS repeat expansion disorder, we cloned GGGGCC repeats, which are the leading genetic cause of C9FTD/ALS. We employed a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors and validated the cloning to facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level using these vectors. In this study, we also differentiated C9FTD/ALS patient-derived induced pluripotent stem cells (iPSCs) to neural stem cells (NSCs) to be used as model systems. The use of iPSCs and NSCs to reveal important insights into the pathogenic mechanisms and to generate multiple neural cell types presents an excellent opportunity for researchers to model neurodegenerative diseases for cell therapy and drug discovery. We further investigated potential nuclear export mechanisms for C9FTD/ALS xtrRNA. The nuclear export mechanisms of xtrRNA in C9FTD/ALS are not well studied. ASOs and siRNAs were employed to knockdown genes of interest to study their involvement in the nuclear export of xtrRNA. We saw promising results on knockdown of TorsinA involved in nuclear export of xtrRNAs, corroborated by a substantial increase in the average number of xtrRNA foci in the nucleus. Our initial study provides evidence that TOR1A may be involved in the nuclear export of aberrant C9FTD/ALS repeat-containing RNAs. Due to the lack of reliable and robust assays to detect RAN translation products, the effect of the knockdown of TorsinA in these cell lines still remains to be explored. But the current study lays the groundwork for a deeper understanding of the less-studied nuclear export mechanisms in C9FTD/ALS and could reveal new therapeutic approaches to selectively block the nuclear export of xtrRNA through the use of RNAi and ASOs. The insights gained from this study will help us understand future events in the xtrRNA life cycle such as repeat translation mechanisms.

Autophagy is a critical cellular maintenance machinery in cells, and prevents the accumulation of toxic protein aggregates, organelles or lipid droplets through degradation via the lysosome. In macro-autophagy, autophagosome first engulfs around aggregates or cellular debris and subsequently fuses with a lysosome that is sufficiently acidic (pH 4.5–5.5), where the contents are then degraded via lysosomal enzymes. Autophagy inhibition as a result of lysosomal acidification dysfunction (pH > 5.5) have been reported to play a major role in various diseases pathogenesis. Hence, there is a pressing need to target lysosomal pH to rescue autophagy. Nanoparticles are attractive materials which has been shown to be efficiently uptaken into cellular organelles and can serve as an agent to specifically localize into lysosomes and modulate its pH. Lipotoxicity, induced by chronic exposure to free fatty acids, and exposure to neurotoxins (e.g. MPP+), elevates lysosomal pH in pancreatic beta cells (Type II Diabetes, T2D) and hepatocytes (Non-alcoholic fatty liver disease, NAFLD), and PC-12 cells (Parkinson’s Disease), respectively. We first tested the lysosome acidification capability of photo-activable nanoparticles (paNPs) and poly (lactic-co-glycolic) acid nanoparticles (PLGA NPs) in a T2D model. Both NPs lowered lysosomal pH in pancreatic beta cells under lipotoxicity and improved insulin secretion function. However, paNPs only release acids upon UV trigger, limiting its applicability in vivo, while PLGA NPs degrade upon lysosome localization. We further showed that PLGA NPs are able to rescue MPP+ induced cell death in a PD model, though it has a slow degradation rate. To attain the most efficacious nanoparticle with a fast degradation and acidification rate, we synthesized acidic nanoparticles (acNPs) based on tetrafluorosuccinic and succinic acids to form optimized nanoparticles. The acNPs showed faster rescue of cellular function compared to PLGA NPs in the PD model. Finally, we tested the acNPs in NAFLD model, and where lysosomal pH reduction by acNPs restored autophagy, reduced lipid accumulation, and improved mitochondria function in high-fat diet mice. In sum, nanoparticles are of potential therapeutic interest for pathologies associated with lysosomal acidity impairment. Future studies include testing the acNPs in NASH disease model and clinical studies. / 2022-05-18T00:00:00Z

Developing microdevices on flexible material attracts scientific community to explore applications in different aspects of health and point of care diagnostics. Flexible substrates offer unique characteristics such as flexibility, stretchability, portability, low-cost, and simple fabrication. Fabrication of cost-effective paper-based analytical devices by wax printing has recently become popular using cellulose filter papers. Paper-based devices need higher temperature to form hydrophobic barrier across paper substrate, rely on large working channels (≥ 500 μm) for liquid handling, and exhibit lower efficiency (~50%) of sample mobility. Such limitations confine applications of wax-based fabrication. In this dissertation, we report printability, fidelity, and applications of wax micropatterns on polyethylene terephthalate-based substrate (PET), which is a a non-cellulosic, non-fibrous, and non-porous material. Resolution, sustainability against heat and biocompatibility was tested on wax micro-features. The patterned devices were explored for variety of applications.First, wax microwells on PET showed mouse embryonic stem cell (mESC) self-renewal or direct differentiation. Second, microfluidic flow was demonstrated on wax printed microchannels on PET which was used to develop distance-based assay. Third, fluidic properties of trinucleotide repeat sequences were investigated on wax microchannels. Fourth, multilayer wax-on-plastic device was fabricated using wax printing with hand painting of conductive materials for electrochemical immunosensing.