Mild Cognitive Impairment is a condition that affects an individual's thinking and decision making capabilities. Specifically, it is one where an individual's capabilities of memorizing, thinking and decision making are less efficient when compared to others. In order to diagnose this condition, a conventional method is to provide the subject with a small challenge they should be completed using pen and paper. This thesis focuses on how this method can be converted to a computer based test. A data visualization tool named Processing has been used to develop a system that runs a game-like test, which is completed by individuals using a mouse. The system then saves the individual's mouse movements in the form of a CSV file. This files are used for further analyzed using JMP Pro on how this data can be used for determining cognitive abilities via computing a metric called Fractal, and what conclusions can be drawn. In order to achieve comparable results, readings from two diverse age groups have been collected. The results using a total of 12 subjects are convincing in that the tool can be used to as a marker for detecting cognitive impairment.

A Doença de Alzheimer (DA) é a principal causa de demência na população idosa e tende a se tornar um grave problema de saúde pública com o aumento da expectativa de vida da população mundial. A perda progressiva de memória, principal sintoma da demência em pacientes com DA, é atribuída a danos sinápticos e à perda neuronal desencadeadas pelo desequilíbrio entre a produção e a depuração do peptídeo A?. Evidências surgidas nos últimos 20 anos apontam os oligômeros solúveis de A? (A?O), produtos de agregação do peptídeo A?, como as principais espécies neurotóxicas na DA. Por conta disso, e também pela ausência de métodos diagnósticos pre-mortem e tratamento eficientes para essa demência, a busca por anticorpos conformacionais específicos para A?O está em ascensão. Testes clínicos com IgG anti-A? resultaram em efeitos colaterais inflamatórios mediados pela porção não variável Fc. Então, anticorpos conformacionais artificiais do tipo scFv, desprovidos de porção Fc, foram selecionados contra A?O. Dentre eles, está NUsc1, que é neuroprotetor contra A?O em cultura primária de neurônios. Neste trabalho, avaliamos a toxicidade de A?Os na linhagem de neuroblastoma humano SH-SY5Y diferenciada em neurônios maduros e comparamos a neuroproteção conferida por diferentes anticorpos contra A?Os, por ensaio de viabilidade celular com MTT. Também avaliamos a especificidade de NUsc1 por A?O comparativamente a lisozima monomérica e oligomérica em ensaio de ELISA, já que outros anticorpos conformacionais reconhecem epítopo compartilhado por estados oligoméricos de outras proteínas amiloidogênicas. Para a validação de células SH-SY5Y como modelo in vitro de neurônios maduros, a diferenciação foi induzida com ácido retinoico e BDNF e as células foram marcadas para as proteínas MAP2 e NeuN em ensaio de imunofluorescência. Células submetidas ao protocolo de diferenciação apresentaram aumento dos níveis dessas proteínas, mudança morfológica condizente com o esperado na maturação neuronal. Posteriormente, o desafio da cultura com A?O indicou morte celular dose-dependente e reversão desta morte segundo a dose administrada dos anticorpos 6E10 e NU-4. Obtivemos um sinal cerca de 400 vezes maior no reconhecimento de A?O por NUsc1 que para oligômeros de lisozima, quando presentes na mesma concentração, indicando forte especificidade de NUsc1 por A?O. Além disso, NUsc1 purificado em sistema de gelfiltração em HPLC não apresenta citotoxicidade em concentração equivalente a dos anticorpos 6E10 e NU-4 em ensaios de neuroproteção em cultura de SH-SY5Y diferenciada, sugerindo que, se NUsc1 for tão eficiente quanto estas IgG\'s, este poderá ser usado em dose não citotóxica. Portanto, podemos concluir que NUsc1 apresenta grande potencial como ferramenta diagnóstica e terapêutica para a DA, mas que mais experimentos para expandir sua validação e potencial ainda são necessários. / Alzheimer\'s Disease (AD) is the leading cause of dementia in the elderly population and tends to become a serious public health problem with increasing life expectancy of the world\'s population. Progressive memory loss, the main symptom of dementia in patients with AD, is attributed to synaptic damage and neuronal loss triggered by imbalance between production and clearance of the A? peptide. Evidence from the last 20 years indicates that soluble A? oligomers (A?O), A? peptide aggregation products, as the main neurotoxic species in AD. Because of this, and also because of the absence of efficient pre-mortem diagnostic and treatment methods for this dementia, the search for conformational antibodies specific for A?O is on the rise. Clinical tests with anti-A? IgG\'s resulted in inflammatory side effects mediated by the non-variable Fc portion. Then, artificial conformational antibodies of the scFv type, lacking the Fc portion, were selected against A?O. Among them is NUsc1, which is neuroprotective against A?O in primary neuronal culture. In this work, we evaluated the toxicity of A?Os in the differentiated SH-SY5Y human neuroblastoma line in mature neurons and compared the neuroprotection conferred by different antibodies against A?O types by MTT cell viability assay. We also evaluated the specificity of NUsc1 for A?O compared to monomeric and oligomeric lysozyme in the ELISA assay, since other conformational antibodies recognize epitope shared by oligomeric states of other amyloidogenic proteins. For the validation of SH-SY5Y cells as an in vitro model of mature neurons, differentiation was induced with retinoic acid and BDNF and the cells were labeled for MAP2 and NeuN proteins in immunofluorescence assay. Cells submitted to the differentiation protocol presented increased levels of these proteins, a morphological change consistent with the expected neuronal maturation. Subsequently, the challenge of culture with A?O indicated dose-dependent cell death and reversion of this death according to the administered dose of 6E10 and NU-4 antibodies. We obtained a 400-fold higher signal in the recognition of A?O by NUsc1 than for lysozyme oligomers, when present at the same concentration, indicating strong specificity of A?O by NUsc1. In addition, NUsc1 purified on HPLC gel-filtration system does not exhibit cytotoxicity at concentration equivalent to 6E10 and NU-4 antibodies in neuroprotection assays in differentiated SH-SY5Y culture, suggesting that, if NUsc1 is as efficient as these IgG\'s, it may be used in a non-cytotoxic dose. Therefore, we can conclude that NUsc1 presents great potential as a diagnostic and therapeutic tool for AD, but that further experiments to expand its validation and potential are still necessary.

A Doença de Alzheimer (DA) é a principal causa de demência na população idosa e tende a se tornar um grave problema de saúde pública com o aumento da expectativa de vida da população mundial. A perda progressiva de memória, principal sintoma da demência em pacientes com DA, é atribuída a danos sinápticos e à perda neuronal desencadeadas pelo desequilíbrio entre a produção e a depuração do peptídeo A?. Evidências surgidas nos últimos 20 anos apontam os oligômeros solúveis de A? (A?O), produtos de agregação do peptídeo A?, como as principais espécies neurotóxicas na DA. Por conta disso, e também pela ausência de métodos diagnósticos pre-mortem e tratamento eficientes para essa demência, a busca por anticorpos conformacionais específicos para A?O está em ascensão. Testes clínicos com IgG anti-A? resultaram em efeitos colaterais inflamatórios mediados pela porção não variável Fc. Então, anticorpos conformacionais artificiais do tipo scFv, desprovidos de porção Fc, foram selecionados contra A?O. Dentre eles, está NUsc1, que é neuroprotetor contra A?O em cultura primária de neurônios. Neste trabalho, avaliamos a toxicidade de A?Os na linhagem de neuroblastoma humano SH-SY5Y diferenciada em neurônios maduros e comparamos a neuroproteção conferida por diferentes anticorpos contra A?Os, por ensaio de viabilidade celular com MTT. Também avaliamos a especificidade de NUsc1 por A?O comparativamente a lisozima monomérica e oligomérica em ensaio de ELISA, já que outros anticorpos conformacionais reconhecem epítopo compartilhado por estados oligoméricos de outras proteínas amiloidogênicas. Para a validação de células SH-SY5Y como modelo in vitro de neurônios maduros, a diferenciação foi induzida com ácido retinoico e BDNF e as células foram marcadas para as proteínas MAP2 e NeuN em ensaio de imunofluorescência. Células submetidas ao protocolo de diferenciação apresentaram aumento dos níveis dessas proteínas, mudança morfológica condizente com o esperado na maturação neuronal. Posteriormente, o desafio da cultura com A?O indicou morte celular dose-dependente e reversão desta morte segundo a dose administrada dos anticorpos 6E10 e NU-4. Obtivemos um sinal cerca de 400 vezes maior no reconhecimento de A?O por NUsc1 que para oligômeros de lisozima, quando presentes na mesma concentração, indicando forte especificidade de NUsc1 por A?O. Além disso, NUsc1 purificado em sistema de gelfiltração em HPLC não apresenta citotoxicidade em concentração equivalente a dos anticorpos 6E10 e NU-4 em ensaios de neuroproteção em cultura de SH-SY5Y diferenciada, sugerindo que, se NUsc1 for tão eficiente quanto estas IgG\'s, este poderá ser usado em dose não citotóxica. Portanto, podemos concluir que NUsc1 apresenta grande potencial como ferramenta diagnóstica e terapêutica para a DA, mas que mais experimentos para expandir sua validação e potencial ainda são necessários. / Alzheimer\'s Disease (AD) is the leading cause of dementia in the elderly population and tends to become a serious public health problem with increasing life expectancy of the world\'s population. Progressive memory loss, the main symptom of dementia in patients with AD, is attributed to synaptic damage and neuronal loss triggered by imbalance between production and clearance of the A? peptide. Evidence from the last 20 years indicates that soluble A? oligomers (A?O), A? peptide aggregation products, as the main neurotoxic species in AD. Because of this, and also because of the absence of efficient pre-mortem diagnostic and treatment methods for this dementia, the search for conformational antibodies specific for A?O is on the rise. Clinical tests with anti-A? IgG\'s resulted in inflammatory side effects mediated by the non-variable Fc portion. Then, artificial conformational antibodies of the scFv type, lacking the Fc portion, were selected against A?O. Among them is NUsc1, which is neuroprotective against A?O in primary neuronal culture. In this work, we evaluated the toxicity of A?Os in the differentiated SH-SY5Y human neuroblastoma line in mature neurons and compared the neuroprotection conferred by different antibodies against A?O types by MTT cell viability assay. We also evaluated the specificity of NUsc1 for A?O compared to monomeric and oligomeric lysozyme in the ELISA assay, since other conformational antibodies recognize epitope shared by oligomeric states of other amyloidogenic proteins. For the validation of SH-SY5Y cells as an in vitro model of mature neurons, differentiation was induced with retinoic acid and BDNF and the cells were labeled for MAP2 and NeuN proteins in immunofluorescence assay. Cells submitted to the differentiation protocol presented increased levels of these proteins, a morphological change consistent with the expected neuronal maturation. Subsequently, the challenge of culture with A?O indicated dose-dependent cell death and reversion of this death according to the administered dose of 6E10 and NU-4 antibodies. We obtained a 400-fold higher signal in the recognition of A?O by NUsc1 than for lysozyme oligomers, when present at the same concentration, indicating strong specificity of A?O by NUsc1. In addition, NUsc1 purified on HPLC gel-filtration system does not exhibit cytotoxicity at concentration equivalent to 6E10 and NU-4 antibodies in neuroprotection assays in differentiated SH-SY5Y culture, suggesting that, if NUsc1 is as efficient as these IgG\'s, it may be used in a non-cytotoxic dose. Therefore, we can conclude that NUsc1 presents great potential as a diagnostic and therapeutic tool for AD, but that further experiments to expand its validation and potential are still necessary.

L’analyse du mécanisme d’interaction de l’héparane sulfate protéoglycane (HSPG) avec lesprotéines amyloïdes et l’effet de paramètres physiologiques (pH,…) sur ce mécanisme nouspermettent une meilleure compréhension des mécanismes menant à l’amyloïdogenèse.La transthyrétine (TTR), molécule circulant à la fois dans le plasma et dans le liquidecéphalo-rachidien, est une des protéines impliquée dans les amyloses. Elle est responsable desamyloses à la TTR et elle joue un rôle dans la maladie d’Alzheimer en séquestrant la protéinebêta-amyloïde (Aβ). La biochromatographie est un outil très efficace pour analyser lemécanisme entre un ligand et son récepteur dans des conditions modulables et se rapprochantdes conditions biologiques. De plus, les nanotubes de carbones (NTCs) peuvent être utiliséspour détecter ou transporter des molécules qui se lient sur leur surface externe et peuventinteragir avec d’autres composés. Au cours de ce travail, un support particulaire a été utilisé où l’HSPG est immobilisé sur desparticules de silice pré-activées par des résidus amines. Ce support remplissant une colonnechromatographique a permis dans un premier temps d’étudier et de comparer les mécanismesd’association entre l’HSPG et une forme sauvage de la TTR et une forme sénile de la TTRextraite d’un patient décédé des suites d’une amylose sénile à la TTR. Cette étude a montréque pour la TTR sauvage, l’association avec l’HSPG est indépendante du pH et implique desinteractions faibles. Pour la TTR sénile, cette association est dépendante du pH. A pH<6,5, laprotonation d’un résidu histidine est observée. De plus, l’étude des paramètresthermodynamiques et de la compensation enthalpie/ entropie montrent un changement dans lemécanisme de fixation avec l’apparition d’interactions ioniques à pH<6,5. Un pH acide estnécessaire pour dissocier et dénaturer partiellement la TTR. L’affinité de la TTR avec l’HSPGdépend de la structure tétramérique quaternaire de la TTR qui présente alors des résiduscapables de créer des interactions. Dans un deuxième temps, cette colonne a permis d’évaluerl’effet de la TTR et du pH sur la liaison Aβ/HSPG. Comme précédemment, la protonationd’un résidu histidine présent sur la Aβ est observée à pH<6,5. Ce résultat confirme des étudesmenées auparavant sur le précurseur de la protéine Aβ. Les résultats thermodynamiques ontmis en évidence que l’affinité de Aβ avec l’HSPG diminuait avec la concentration croissanteen TTR et l’étude des chromatogrammes associés a montré que la TTR ne séquestrait passeulement Aβ mais la clivait en fragments plus courts qui diminuent son affinité avecl’HSPG. Dans la maladie d’Alzheimer, la TTR exerce une activité protéolytique vis-à-vis deAβ.2. Dans un troisième temps, l’effet de la fonctionnalisation de la TTR par des nanotubes decarbone sur la liaison TTR/HSPG a été étudié. Les résultats obtenus montrent que lesinteractions entre l’HSPG et la TTR-NTC sont de type van der Waals et hydrogène. Lesparamètres thermodynamiques des liaisons TTR/HSPG et TTR-NTC/HSPG sont similairespour des pH>6. A pH<6, il n’existe quasiment pas de différences entre les valeurs obtenues àpH >6 et celles obtenues à pH<6. Les NTCs empêcheraient la formation de liaisons ioniques / The analysis of the heparan sulfate proteoglycan (HSPG) association mechanism withamyloid proteins and the effect of physiological parameters (pH,…) on this mechanism allowa better understanding of the mechanisms leading to amyloidogenesis.Transthyretin (TTR), which circulates in both plasma and cerebrospinal fluid, is one of theproteins involved in amyloidosis. It leads to TTR amyloidosis and plays a role in Alzheimer’sdisease in sequestrating the beta-amyloid (Aβ) protein. Biochromatography is an effectivetool for the analysis of the mechanism involved between a ligand and its receptor inadjustable conditions which could be close to biological conditions. Moreover, carbonnanotubes can be used to detect or to carry molecules which bind on its external surface andcould interact with other molecules. In this work, a particulate support was used where the HSPG was immobilized on the silica particles preactivated by amine residues. This support filling a column was used to study andcompare association mechanisms between HSPG and a wild type TTR form and a senile formof the TTR which was was extracted from a patient who died of cardiac failure with a senilesystemic amyloid. This study showed that the association between wild type TTR and HSPGwas independent of the pH and involved weak interactions.For the senile TTR, this association was dependent on pH. At pH<6,5, a histidine residue wasprotonated. Moreover, the study of both thermodynamical parameters and enthalpy-entropycompensation showed a change in the association mechanism with involvement of more ionicinteractions at pH<6,5. An acidic pH was necessary to dissociate and partially denaturate theTTR. The affinity of the TTR with HSPG depends on the tetrameric quaternary structure ofthe TTR which presents some residues which are able to create interactions.In a second time, this column was used to evaluate the effect of both the TTR and the pH onAβ/HSPG binding. As previously, the protonation of a histidine residue present on Aβ wasobserved at pH<6,5. This result confirmed studies on the Aβ precursor. Thermodynamicalvalues highlighted that the affinity of Aβ for l’HSPG decreased with the increase of TTRconcentration, and the study of the chromatograms associated showed that TTR sequestratedAβ and cut Aβ in smaller fragments which decreased its affinity for HSPG. In Alzheimer’sdisease, TTR has a proteolytic activity on Aβ.In a third time, the effect of the carbon nanotubes (CNTs), immobilized on TTR, on theTTR/HSPG binding was studied. Results showed that interactions between TTR-NTC andHSPG are van der Waals and hydrogen. Thermodynamical data of TTR/HSPG et TTRNTC/HSPG bindings are similar at pH>6. At pH<6, no differences between values obtainedat all pH values for TTR-NTC. NTCs would avoid ionic interactions formations.