Intact white matter is critical for normal cognitive function. In traumatic brain injury (TBI), chronic cerebral hypoperfusion and Alzheimer’s disease (AD) damage to white matter is associated with cognitive impairment. However, these conditions are associated with grey matter damage or with other pathological states and the contribution of white matter damage in isolation to their pathogenesis is not known. Furthermore, TBI is a risk factor for AD and cerebral hypoperfusion is an early feature of AD. It is hypothesised that white matter damage following TBI or chronic cerebral hypoperfusion will be associated with cognitive deficits and that white matter changes after injury contribute to AD pathogenesis. To investigate this, this thesis examined the contribution of white matter damage to cognitive deficits after TBI and chronic cerebral hypoperfusion and furthermore, investigated the role of white matter damage in the relationship between TBI and AD. Three studies addressed these aims. In the first, mild TBI was induced in wild-type mice and the effects on axons, myelin and neuronal cell bodies examined at time points from 4 hours to 6 weeks after injury. Spatial reference learning and memory was tested at 3 and 6 weeks after injury. Injured mice showed axonal damage in the cingulum, close to the injury site in the hours after injury and at 6 weeks, damage in the thalamus and external capsule were apparent. Injured and sham animals had comparable levels of neuronal damage and no change was observed in myelin. Injured animals showed impaired spatial reference learning at 3 weeks after injury, demonstrating that selective axonal damage is sufficient to impair cognition. In the second study mild TBI was induced in a transgenic mouse model of AD and the effects on white matter pathology and AD-related proteins examined 24 hours after injury. There was a significant increase in axonal damage in the cingulum and external capsule and parallel accumulations of amyloid were observed in these regions. There were no changes in tau or in overall levels of AD-related proteins. This suggests that axonal damage may have a role in mediating the link between TBI and AD. The third study used a model of chronic cerebral hypoperfusion in wild type mice and investigated white matter changes after one and two months of hypoperfusion as well as a comprehensive assessment of learning and memory. Chronic cerebral hypoperfusion resulted in diffuse myelin damage in the absence of ischaemic neuronal damage at both 1 and 2 months after induction of hypoperfusion. Hypoperfused animals also showed minimal axonal damage and microglial activation. Cognitive testing revealed a selective impairment in spatial working memory but not spatial reference or episodic memory in hypoperfused animals, showing that modest reductions in blood flow have effects on white matter sufficient to cause cognitive impairment. These results demonstrate that selective damage to white matter components can have a long-term impact on cognitive function as well as on the development of AD. This suggests that minimisation of axonal damage after TBI is a target for reducing subsequent risk of AD and that repair or prevention of white matter damage is a promising strategy for rescuing cognitive function in individuals who have experienced mild TBI or chronic cerebral hypoperfusion.

La maladie d’Alzheimer est une maladie neurodégénérative, elle représente 70% des formes de démences et affecte près de 860 000 personnes en France. Cette maladie est caractérisée par deux lésions neuropathologiques : les Dégénérescences neurofibrillaires et les Plaques séniles. Ces dernières sont principalement constituées de peptides amyloïdes (A&#946;) résultant du clivage d’une protéine membranaire appelée Précurseur du peptide amyloïde (APP). L’étude des formes familiales monogéniques a montré que des mutations des gènes de l’APP et des Présénilines 1 et 2 conduisaient systématiquement à une augmentation de la production d’A&#946;. Cette observation a permis l’élaboration de la cascade amyloïde plaçant le métabolisme de l’APP au centre du processus physiopathologique. Même si aujourd’hui ce métabolisme commence à être relativement bien connu, plusieurs zones d’ombres subsistent encore. Dans l’optique de caractériser de nouveaux acteurs intervenant dans ce métabolisme, nous avons émis une hypothèse qui repose sur deux constatations : (i) les protéines impliquées dans l’étiologie de la maladie sont différentiellement exprimées entre les cerveaux des patients et ceux des témoins (ii) dans le cerveau, de nombreuses métalloprotéases participent aux même mécanismes que l’APP (adhésion cellulaire, neuroinflammation, plasticité neuronale...), certaines sont aussi directement actrices du métabolisme de l’APP en tant qu’&#945;-sécrétase (ADAM9, ADAM10 et ADAM17) ou en dégradant l’A&#946; (NEP, IDE, MMP2, MMP3 et MMP9). Nous avons donc supposé que les métalloprotéases présentant une différence d’expression entre le tissu cérébral des malades et celui des témoins soient des candidates intéressantes pour moduler le métabolisme et le trafic de l’APP. Une première analyse transcriptomique par biopuce, à partir d’ARN totaux issus des cerveaux de 12 malades et de 12 témoins, nous a permis d’identifier quatre métalloprotéases présentant une différence d’expression significative (p<10-5) : ADAMTS16, ADAM17, ADAM30 et ADAM33. Nous avons cherché à confirmer ce résultat par une autre technologie sur un plus grand nombre d’échantillons (malades n=52 et témoins n=42). Seules ADAM30 et ADAM33 ont pu être validées. Nous avons également pu observer que l’expression d’ADAM30 dans le tissu cérébral des malades est inversement proportionnelle à la quantité d’A&#946;42 déposée dans la parenchyme (A&#946;42 la forme d’A&#946; la plus neurotoxique). De plus, au niveau cérébral, l’expression d’ADAM30 est restreinte aux neurones, cellules sièges du métabolisme de l’APP. Nous avons donc sélectionné ADAM30 comme intervenante potentielle dans le métabolisme de l’APP. Pour tester notre hypothèse, nous avons sous- et sur-exprimé ADAM30 dans deux modèles cellulaires différents. Nous avons mis en évidence que la sur-expression d’ADAM30 entraîne une diminution de l’ensemble des produits du métabolisme de l’APP. En mutant le site catalytique de cette protéase, nous avons remarqué que cette action sur le métabolisme de l’APP est dépendante de cette activité catalytique. De manière cohérente, une sous-expression d’ADAM30 entraîne une augmentation de l’ensemble des produits du métabolisme de l’APP. En utilisant les inhibiteurs alcalisant, nous avons démontré que l’effet d’ADAM30 sur le métabolisme de l’APP met en jeu la dégradation par le lysosome. Des expériences d’immunofluorescence ont attesté qu’ADAM30 est localisée dans le réticulum endoplasmique et l’appareil de Golgi et qu’elle co-localise fortement avec l’APP dans ces organites. Au vu des résultats obtenus durant ces quatre années, nous pensons qu’ADAM30 pourrait être une protéine clé de la régulation de l’APP en inhibant son acheminement jusqu’à la membrane plasmique et en favorisant sa dégradation par le lysosome. [...] / Alzheimer’s disease (AD) is the most common neurodegenerative disorder of the old age, characterized by the presence of two major neuropathological features : neurofibrillary tangles and senile plaques. These plaques are composed of the A&#946; peptides cleavage product of the amyloid precursor protein (APP). Proteolytic processing of APP is modulated by the action of enzymes &#945;-, &#946;- and &#947;-secretases with the latter two mediating the amyloidogenic pathway. Suggesting that processing of APP is a key step in the pathology of AD. However, even if extensively studied, this APP metabolism is still not fully characterized. With this background, we postulate that the characterization of new actors of the APP metabolism might help for a more subtle understanding of this APP metabolism and trafficking. We focused on the ADAMs and related proteins with the hypothesis that ADAMs and related proteins, under- or over-expressed in the brain of AD cases compared with the one of controls, may be of particular interest. Beyond the obvious implication of several ADAMs as &#945;-secretases, this hypothesis was also driven by several observations : (i) ADAMs have been involved in numerous biological processes including brain development, plasticity and repair as APP; (ii) several metalloproteases (MMP-2, -3 and -9) have been described to degrade A&#946; peptides. Using microarray to screen the expression of 117 ADAMs and MMPs was analyzed using total RNA extracted from cerebral tissue of 12 AD cases and 12 controls. We observed that 4 ADAMs were differentially expressed. We first confirmed that the ADAM30 expression was decreased in AD brains and we observed that ADAM30 under-expression was correlated with an increase in A&#946;42 deposition in AD brains. Consistently, over-expression of ADAM30 led to decrease APP metabolism and as a consequence, A&#946; secretion in two different cell lines (Moreover, under-expressed ADAM30 increases APP processing and A&#946; generation). This modification of the APP metabolism was directly linked to the ADAM30 catalytic properties. Our data suggest that catalytic activity of ADAM30 takes an important place in APP processing in a lysosome dependent manner and AD pathophysiological process.

Neuropsychological testing is one method used in the diagnosis of Alzheimer’s disease and other cognitive disorders. However, the testing process may be affected by subtle external factors which if not controlled for may have the ability to affect the scores obtained. The primary aim of this thesis was to investigate the effects of some of these external factors, namely caffeine, non-oily fish consumption and time of day. A secondary aim was to evaluate the use of a novel virtual assessment as a possible tool for the early detection of AD. Healthy elderly participants over the age of sixty with no existing cognitive impairment or neurological condition were recruited to take part. For each external factor investigated participants were required to undertake a cognitive assessment. The results demonstrated that subtle external factors present during a typical testing session have the ability to significantly affect the scores obtained. Scores on one part of the virtual test correlated with existing tests used for the early detection of cognitive impairment and were significantly lower in participants classified as mildly impaired. With further modification this test has the potential to be used as an early detection tool. The results have implications for the interpretation of neuropsychological test scores which may be considered when classifying participants, determining treatment interventions, selecting participants for research and making a diagnosis. These findings have important considerations for psychological and cognitive research that investigates human brain function.

La maladie d'Alzheimer (MA) est une maladie neurodégénérative irréversible et progressive entraînant des troubles cognitifs et comportementaux. L'inflammation est caractéristique de la MA. L'interleukine-10 (IL-10), une cytokine anti-inflammatoire a été associée à un risque plus faible de développer la MA. Cependant le lien entre l'IL-10 et la progression de la MA n'a jamais été étudié. Le but de cette thèse a été d'étudier le rôle de l'IL-10 dans le développement ainsi que dans la progression de la MA. Pour mener à bien cette étude, 31 sujets atteints par la MA et 20 sujets contrôles cognitivement intacts ont été recrutés. En fonction de la vitesse de diminution du test de Mini-Mental State Examination et de l’évolution des troubles cognitifs sur deux ans, les patients souffrant de la MA ont été divisés en deux sous-groupes: les patients avec une progression lente (MA lent) et ceux avec une progression rapide (MA rapide). Les analyses se sont portées sur la concentration d’IL-10 en périphérie (plasma, production par les cellules mononuclées du sang périphérique (PBMCs) après stimulation par les peptides Aβ) ainsi que le polymorphisme de son promoteur en position -592, -819 et -1082. En complément, d’autres cytokines impliquées dans l’inflammation ont été étudiées : l’IL-6 (sa concentration plasmatique, sa production par les PBMCs à la suite d’une stimulation par les peptides Aβ et son polymorphisme en position -174) et les polymorphismes du TGF-β1 (-10 et - 25), de l’IFN-γ (-874) et du TNF-α (-308) ainsi que le gène de l'apolipoprotéine E (ApoE). Une étude de la longueur des télomères, liée à l’inflammation, a été aussi réalisée. Les résultats ont montré une association entre le génotype AA et l’allèle A du polymorphisme de l’IFN-γ en position -874 avec la progression rapide de la MA. Une augmentation statistiquement significative de la production d'IL-10 après stimulation par les peptides Aβ a été montrée chez les patients atteints avec une progression lente (MA lent). Une longueur significativement plus courte des télomères a été aussi associée aux patients MA lent. L’ensemble de ces travaux suggère qu’un profil de forte production de l’IL-10 ainsi qu’un profil génétique d’IFN-γ (TT -874) pourrait ralentir la progression de la MA. Il est aussi apparu que la longueur des télomères pourrait être un marqueur du déficit cognitif. Il est clair que ces résultats préliminaires ont besoin d’être confirmés par une étude de plus grande envergure, avec un nombre de patients plus élevé. / Alzheimer’s disease (AD) is an irreversible and progressive neurodegenerative disorder leading to cognitive and behavioral impairment. Inflammation is hallmark of AD although the exact mechanisms involved and the roles of the different inflammatory components are far less clear. Interleukin-10 (IL-10) is a key anti-inflammatory cytokine and IL-10 -1082 A > G polymorphism has been associated with a lower risk of developing AD although the link between IL-10 and the AD progression have never been studied. The aim of this study is to study the role of IL-10 in the risk of developing AD and its role in AD progression. In order to complete successfully this study, 31 AD patients and 20 cognitively intact controls were recruited. Depending of the rate of decrease of mini-mental state test examination (MMSE) and evolution of cognitive disorders, AD patients were divided in two subgroups: patients with slow progression (AD slow) and those with fast progression (AD fast). Analysis were focused on periphery concentration of IL-10 (plasma and and its production by peripheral blood mononuclear cells (PBMCs) after Aβ peptides stimulation) as well as its promoter polymorphism in position -592, -819 and -1082. In addition, other cytokines involved in inflammation were studied: IL-6 (its plasma concentration, its production by PBMCs following stimulation with Aβ peptides and its polymorphism at position -174) and polymorphisms of TGF-β1 (-10 to - 25), IFN-γ (-874) and TNF-α (-308) as well as the gene polymorphisms of Apolipoprotein E (ApoE). A study of telomere length, link to inflammation, was also performed. Results showed IFNγ -874AA genotype and -874A allele was associated with AD fast progression. A statistically significant increase of IL-10 production by PBMCs stimulated with Aβ peptides was shown in AD slow patients. A significantly shorter telomere length was also associated with AD slow patients. All of this work suggests that a profile with high IL-10 production and high IFN-γ (-874 TT) genotype could confer a slower AD progression. It was also found that telomere length may be a marker of cognitive impairment. It is clear that these preliminary results need to be confirmed in a larger study with a larger number of patients.

INTRODUCTION. Les troubles neurocognitifs sévères ou démences sont notamment définis par la CIM-10 et le DSM-5. Ils englobent un cadre nosographique large : démence d’Alzheimer, démence vasculaire, démence à corps de Lewy, dégénérescence lobaire fronto-temporale, etc. Chaque type de démence possède des critères diagnostiques propres et des facteurs de risque partiellement identifiés. Identifier les troubles cognitifs dans les grandes bases de données est une question complexe, qui doit tenir compte de l’évolution des connaissances. Notre premier objectif était de décrire l’évolution des codages de démences dans la base nationale du Programme de Médicalisation des Systèmes d’Information (PMSI) de court séjour, au fil de l’évolution des critères diagnostiques. Notre deuxième objectif était d’énumérer les principaux facteurs associés connus de maladie d’Alzheimer. Notre troisième objectif était de déterminer les facteurs associés à l’apparition d’une maladie d’Alzheimer dans la base nationale du PMSI de court séjour.METHODES. Pour le premier travail, nous avons utilisé les diagnostics principaux sur le site ScanSanté pour le PMSI de court séjour de 2007 à 2017. Pour le deuxième travail, nous avons effectué une synthèse des revues de littérature et méta-analyses en utilisant les moteurs de recherche PubMed et LiSSa. Pour le troisième travail, nous avons réalisé une étude analytique par fouille de données dans la base nationale du PMSI de court séjour chez les patients âgés de 55 ans ou plus en 2014 : nous avons sélectionné 137 variables explicatives potentielles en 2008 ; la variable à expliquer était la maladie ou démence d’Alzheimer en 2014.RESULTATS. Notre premier travail sur l’identification des démences met en évidence une diminution des séjours hospitaliers avec pour diagnostic principal une maladie ou démence d’Alzheimer, avec un glissement vers d’autres troubles mentaux organiques ; une stabilité des séjours hospitaliers avec pour diagnostic principal une démence vasculaire mais avec une modification des sous-diagnostics (diminution des diagnostics principaux d’infarctus multiples et augmentation de tous les autres sous-types) ; une augmentation importante des séjours hospitaliers avec pour diagnostic principal une démence ou autre trouble cognitif persistant ou tardif liés à la consommation d’alcool ; une évolution homogène sur l’ensemble du territoire français. Ces résultats sont en faveur d’un codage respectueux des évolutions de la littérature. Nos deux travaux suivants sur l’identification des populations à risque permettent d’identifier plusieurs facteurs associés à la maladie ou démence d’Alzheimer, notamment l’âge, le sexe féminin, le diabète de type 2, la dépression, la dénutrition, les troubles bipolaires, psychotiques et anxieux, le faible niveau de scolarité, l’excès d’alcool, l’épilepsie, les chutes après 75 ans et l’hypertension intracrânienne. Ces facteurs associés peuvent être des facteurs de risque, des symptômes précoces, révélateurs ou précipitants.CONCLUSION. Identifier les troubles cognitifs dans les grandes bases de données implique de bien comprendre l’évolution des codages de démence, qui semble respecter l’évolution des connaissances. L’identification des patients ayant des facteurs associés aux démences permet un repérage précoce plus ciblé, puis une bonne identification du diagnostic étiologique nécessaire à une prise en charge adaptée. / INTRODUCTION. Severe neurocognitive disorders or dementias are defined by ICD-10 and DSM-5. They encompass a broad nosographic framework: Alzheimer's dementia, vascular dementia, Lewy body dementia, frontal-temporal lobar degeneration, etc. Each type of dementia has its own diagnostic criteria and partially identified risk factors. Identifying cognitive disorders in large databases is a complex issue, which must take into account changes in knowledge. Our first objective was to describe the evolution of dementia coding in the national database of the Medicalization of Information Systems Program (PMSI) for short stays, as diagnostic criteria evolved. Our second objective was to summarize the main known associated factors of Alzheimer's disease. Our third objective was to determine the factors associated with the onset of Alzheimer's disease in the national database of the short stay PMSI.METHODS. For the first work, we used the main diagnoses on the ScanSanté site for the short stay PMSI from 2007 to 2017. For the second work, we synthesized the literature reviews and meta-analyses using the PubMed and LiSSa search engines. For the third work, we conducted an analytical study by data mining in the national database of the short stay PMSI for patients aged 55 years or older in 2014: we selected 137 potential explanatory variables in 2008; the dependant variable was Alzheimer's disease or dementia in 2014.RESULTS. Our first work on the identification of dementias shows a decrease in inpatient stays with a main diagnosis of Alzheimer's disease or dementia, with a shift towards other organic mental disorders; stability of inpatint stays with a main diagnosis of vascular dementia but with a modification of under-diagnosis (decrease in main diagnoses of multiple heart attacks and increase in all other subtypes); a significant increase in inpatient stays with a main diagnosis of dementia or other persistent or late cognitive disorders related to alcohol consumption; a homogeneous evolution throughout the French territory. These results support a coding that respects the evolution of the literature. Our next two studies on the identification of at-risk populations identify several factors associated with Alzheimer's disease or dementia, including age, gender, diabetes mellitus, depression, undernutrition, bipolar, psychotic and anxiety disorders, low education, excess alcohol, epilepsy, falls after age 75 and intracranial hypertension. These associated factors may be risk factors, early, revealing or precipitating symptoms.CONCLUSION. Identifying cognitive disorders in large databases requires a good understanding of the evolution of dementia coding, which seems to respect the evolution of knowledge. The identification of patients with factors associated with dementia allows a more focused early identification and then proper identification of the etiological diagnosis necessary for appropriate management.

Bakgrund Personer med demenssjukdom har stor risk att utveckla beteendemässiga och psykiska symtom efter kirurgi vilket ställer höga krav på omvårdnadsarbetet i det postoperativa förloppet. Aktuell forskning som undersöker sjuksköterskor som har hand om denna patientgrupp i det postoperativa förloppet är begränsad och med anledning med detta ansågs det värdefullt att genomföra en studie som undersöker detta fenomen närmare. Syfte Studien syftade till att undersöka sjuksköterskors kunskaper om demenssjukdom samt beskriva sjuksköterskors arbetssätt i att lindra beteendemässiga och psykiska symtom hos personer med demenssjukdom som genomgått kirurgi. Metod Detta var en deskriptiv tvärsnittsstudie vilken använde sig av en onlinebaserad enkätundersökning som datainsamlingsmetod. Instrumentet Basic Knowledge of Alzheimer’s Disease (BKAD) användes för att testa kunskaper om den vanligaste typen av demenssjukdom. Kunskapen om beteendemässiga och psykiska symtom vid demens och metoder som sjuksköterskor använder sig av för att lindra dessa undersöktes genom utformade frågeställningar. Data inhämtades från januari till april månad år 2019 och totalt deltog 50 sjuksköterskor. Resultat Kunskapen om Alzheimers sjukdom var bristande och likaså även kunskapen om hur man behandlar beteendemässiga och psykiska symtom vid demens. Sjuksköterskors möten med denna patientgrupp rankades överlag som svåra och det visade sig att sjuksköterskor hade haft flertalet sådana möten i den postoperativa vårdverksamheten. Vidare rapporterades det att sjuksköterskors utbildning i demensvård från den grundläggande sjuksköterskeutbildningen var minimal och att utbildning i demensvård borde höjas i den grundläggande sjuksköterskeutbildningen. Slutsats och kliniska implikationer Demensvård som läroämne bör få större omfattning i den grundläggande sjuksköterskeutbildningen. Utbildningsinsatser bör prioriteras för att höja sjuksköterskors kunskaper om vård för personer med demenssjukdom genom organisatoriskt och pedagogiskt stöd. Om kunskapen i demensvård höjs torde även omvårdnaden för personer med demenssjukdom bli bättre. / Background People with dementia have a great risk of developing behavioral and psychological symptoms after surgery. This puts challenges on the nurses’ care for these patients. Current research that investigate nurses’ who care for these individuals in the postoperative phase is limited. Therefore, it was considered being important to investigate this phenomenon further.    Aim The study sought to investigate nurses’ knowledge of dementia, and their work methods in alleviating behavioral and psychological symptoms in dementia for people who had undergone surgery. Method The study was carried out as a descriptive, cross-sectional study using online surveys. The Basic Knowledge of Alzheimer’s Disease (BKAD) was used to test participants’ knowledge of Alzheimer’s disease. The knowledge of behavioral and psychological symptoms in dementia, and nurses’ working methods to alleviate these behaviors and symptoms was investigated by using developed questions. The study collected data from January to April, 2019 and a total of 50 nurses’ participated.  Result There was a deficit in the knowledge of Alzheimer’s disease, and also in the knowledge of how to treat behavioral and psychological symptoms in dementia. Nurses’ reported an overall difficulty in treating a person suffering from behavioral and psychological symptoms, and nurses’ also reported having several encounters with a person with dementia in the surgical ward. Nurses’ stated that training in dementia care in the baccalaureate program was low, and for this reason nurses’ stated that they wished that dementia care would get more attention. Conclusion and clinical implication Dementia care in the baccalaureate program would benefit from getting more attention. Different interventions in education of dementia care by using organizational support ought to be implemented in this process. If the knowledge of dementia care is increased there is a chance that the care for these individuals would become better.

Introduktion: Beteendemässiga och psykiska symptom (BPSD) är vanligt förekommande hos personer med Alzheimers sjukdom. Det påverkar den enskilde, närstående och vårdgivare negativt. Läkemedelsbehandling ska inte ses som ett förstahandsalternativ utan fokus ska istället ligga på omvårdnadsinterventioner. Syftet var att beskriva omvårdnadsinterventioner som kan lindra beteendemässiga och psykiska symptom hos personer med Alzheimers sjukdom. Metoden var litteraturstudie där tio artiklar inkluderades efter kritisk granskning. Artiklarnas innehåll analyserades och sex kategorier med omvårdnadsinterventioner bildades: musikterapi, fysisk aktivitet, ljusterapi, kognitiv rehabilitering, terapeutiska samtal samt djurterapi. Resultatet visade att det fanns omvårdnadsinterventioner som påverkade BPSD för stunden men inget som varade över tid. Slutsats: Studien visar att det finns begränsat med forskning där omvårdnadsinterventioner ger god effekt på BPSD hos personer med Alzheimers sjukdom. / Introduction: Behavioral and Psychological Symptoms of Dementia (BPSD) are common in people with Alzheimer’s disease. This affects the individual, family members and caregivers adversely. Drug treatment should not be seen as a first choice, but the focus should instead be on nursing interventions. The aim was to describe nursing interventions that can alleviate behavioral and psychological symptoms in people with Alzheimer's disease. The method was a literature review in which ten articles after critical review were included. The contents of the articles were analyzed and six categories of nursing interventions were formed: music therapy, physical activity, light therapy, cognitive rehabilitation, therapeutic conversation and animal assisted activity. The results showed that there were nursing interventions that affected BPSD for the moment but nothing that lasted over time. Conclusion: The study shows that there is limited research in which nursing interventions gives good effect on BPSD in people with Alzheimer's disease.

A doença de Alzheimer é uma doença neurodegenerativa que causa perdas de memória, danos cognitivos e no comportamento, sendo considerada uma das causas principais de demência entre a população. Esta doença é caracterizada por uma intensa perda neuronal colinérgica, de forma que tanto os inibidores da enzima acetilcolinesterase (AChE) quanto compostos antioxidantes possam ser empregados como neuroprotetores. Nesse sentido, plantas da família Lycopodiaceae vêm sendo estudadas como fonte de novos alcalóides anticolinesterásicos desde a descoberta da huperzina A, isolada a partir da planta chinesa Huperzia serrata. Este trabalho foi realizado com o objetivo de caracterizar e isolar os alcalóides majoritários de Huperzia acerosa, H. heterocarpon, H. quadrifariata, H. reflexa, Lycopodiella cernua, Lycopodium clavatum e L. thyoides, de ocorrência no estado do Rio Grande do Sul, bem como avaliar as atividades anticolinesterásicas e antioxidantes in vitro e in vivo para os extratos. Extratos de alcalóides totais de espécies de L. clavatum e L. thyoides ocorrentes no Brasil e Argentina foram comparados quanto ao seu conteúdo químico, aliado à atividade anticolinesterásica. A atividade inibitória para AChE e butirilcolinesterase (BuChE) dos alcalóides isolados também foi ensaiada, juntamente com a determinação das propriedades citotóxicas exercidas pelos extratos sobre uma linhagem de gliomas C6. Todos os extratos de alcalóides foram analisados através de CG-EM, sendo os perfis de fragmentação dos compostos comparados através de dados da literatura. L. clavatum e L. thyoides possuem perfil químico similar, com licopodina e acetildiidrolicopodina como alcalóides principais, enquanto que L. cernua possui apenas dois alcalóides, cernuína e licocernuína. Os extratos de alcalóides de H. acerosa, H. quadrifariata e H. reflexa também foram analisados, sendo detectados tanto compostos do grupo licopodano quanto do grupo flabelidano, juntamente com outros alcalóides cujas estruturas ainda são desconhecidas. Os alcalóides encontrados em H. heterocarpon não puderam ser elucidados a partir de dados de fragmentação, uma vez que eles não coincidiram com nenhum perfil da literatura. Nesse estudo verificamos que dentre os extratos analisados, H. quadrifariata e H. reflexa promoveram a maior inibição para a enzima AChE obtida de eritrócitos humanos, com IC50 de 2,0 e 0,11 μg/mL, respectivamente, enquanto que H. heterocarpon foi o único extrato com maior seletividade para a BuChE (IC50 = 8,3 μg/mL). Além disso, dentre os alcalóides isolados licopodina, acetildiidrolicopodina, cernuína, licocernuína e clavolonina, apenas acetildiidrolicopodina e cernuína possuem inibição importante, sendo que nenhum deles possui efeito butirilcolinesterásico significativo. Também foi estimada a atividade anticolinesterásica para os extratos de alcalóides totais de L. clavatum e L. thyoides empregando-se homogenatos de córtex, hipocampo e estriato de ratos como fonte enzimática, sendo determinadas suas curvas de inibição com distintos tempos de incubação em uma faixa de concentrações. Também foi verificada para os dois extratos uma inibição do tipo competitiva/não-competitiva, bem como seu perfil antioxidante in vitro pelos métodos de descoloração do radical DPPH, degradação da 2-deoxirribose, TRAP e óxido nítrico. Após um tratamento agudo em camundongos de 14 meses com os extratos, foram verificados os efeitos antioxidantes através do método de TBA-RS, e para as enzimas catalase e superóxido dismutase. Do mesmo modo, verificamos que os mesmos extratos promoveram uma diminuição na atividade da acetilcolinesterase, quando administrados por via intraperitoneal. Nessa metodologia, os extratos mais potentes foram H. quadrifariata e H. reflexa, corroborando o efeito in vitro encontrado para os mesmos. Os extratos purificados de alcalóides de L. clavatum e L. thyoides com habitat no Brasil e Argentina foram comparados quanto ao seu perfil químico e biológico. Nesse estudo, foi verificado que os alcalóides licopodina e acetildiidrolicopodina são os compostos majoritários para todas as espécies, tendo L. clavatum maior atividade anticolinesterásica em relação a L. thyoides, tanto a espécie brasileira quanto a espécie argentina. / Alzheimer’s disease is a neurodegenerative disease which causes memory loss, cognitive and behavioral damages, considered to be the leading cause of dementia among the elderly. This disease is characterized for an intense cholinergic loss, and the use of acetylcholinesterase (AChE) inhibitors together with antioxidant compounds as neurprotectors is a strategy for the treatment. In this sense, Lycopodiaceae plants are well studied as a source of new anticholinesterasic alkaloids since the discovery of huperzine A, isolated from Chinese Huperzia serrata. This work was conducted with the objective of characterize and isolate the main alkaloids from Huperzia acerosa, H. heterocarpon, H. quadrifariata, H. reflexa, Lycopodiella cernua, Lycopodium clavatum and L. thyoides, with occurrence in Rio Grande do Sul state, and also evaluate the anticholinesterasic and antioxidant activities in vitro and in vivo for the extracts. Alkaloidal extracts from L. clavatum and L. thyoides species with habitat in Brazil and Argentina were compared to their chemical content, together with their anticholinesterasic activity. The inhibitory effect for AChE and butyrylcholinesterase (BuChE) for the isolated alkaloids was also evaluated, together with the determination of the cytotoxic properties exherced by the extracts in a gliome C6 cell line. Every alkaloidal extract was analyzed by means of GC-MS, and the fragmentation patterns for the compounds were compared with those from literature. L. clavatum and L. thyoides have similar chemical profile, with lycopodine and acetyldihydrolycopodine as main alkaloids, while L. cernua has only two alkaloids, cernuine and lycocernuine. The alkaloidal extracts of H. acerosa, H. quadrifariata e H. reflexa were also analyzed, and compounds from lycopodane and flabellidane groups were detected, along with other alkaloids whose structures are still unknown. The alkaloids found in H. heterocarpon could not be elucidated with fragmentation dates so far, since they did not coincide with those found in literature. We also verified that among the extracts, H. quadrifariata and H. reflexa promoted a higher inhibition for AChE obtained from human erythrocytes, with a IC50 value of 2,0 e 0,11 μg/mL, respectively, while H. heterocarpon extract was the only more selective for BuChE (IC50 = 8,3 μg/mL). Moreover, among the isolated alkaloid lycopodine, acetyldihydrolycopodine, cernuine, lycocernuine and clavolonine, only acetyldihydrolycopodine and cernuine possess an important inhibition, and none significantly inhibited butyrylcholinesterase. It was also assessed the anticholinesterase effect for the alkaloidal extracts of L. clavatum and L. thyoides employing cortex, hippocampus and striatum rat brain homogenates as enzymatic sources, being determined the incubation time-inhibition curves over a concentrations range. It was also verified for both extracts an inhibitory effect of the competitive/non competitive mixed type, as well as their in vitro antioxidant profile using the methods of DPPH radical decoloration, 2-deoxyrribose degradation, TRAP and nitric oxide. After an acute treatment in 14 months-aged mice, we verified the antioxidant effects though TBA-RS and also the enzymes catalase and superoxide dismutase. In the same way, we found out that the same extracts reduced the acetylcholinesterase activity when administered by intraperitoneal injection. Using this methodology, the most potent extracts were H. quadrifariata and H. reflexa, corroborating the in vitro effect found for them. The purified alkaloidal extracts of L. clavatum and L. thyoides with habitat in Brazil and Argentine were compared to their chemical and biological profile. In this study, we found out that lycopodine and acetyldihydrolycopodine are the main alkaloids found for all species, and L. clavatum possesses a better inhibition against AChE when compared to L. thyoides for both Brazilian and Argentinean collected species.

Dementia is a clinical state caused by neurodegeneration and characterized by a loss of function in cognitive domains and behavior. Alzheimer’s disease (AD) is the most common form of dementia. Although the amyloid β (Aβ) protein and hyperphosphorylated tau aggregates in the brain are considered to be the key pathological hallmarks of AD, the exact cause of AD is yet to be identified. In addition, clinical diagnoses of AD can be error prone. Many previous studies have compared the clinical diagnosis of AD against the gold standard of autopsy confirmation and shown substantial AD misdiagnosis Hippocampal sclerosis of aging (HS-Aging) is one type of dementia that is often clinically misdiagnosed as AD. AD and HS-Aging are controlled by different genetic architectures. Familial AD, which often occurs early in life, is linked to mainly mutations in three genes: APP, PSEN1, and PSEN2. Late-onset AD (LOAD) is strongly associated with the ε4 allele of apolipoprotein E (APOE) gene. In addition to the APOE gene, genome-wide association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs) in or close to some genes associated with LOAD. On the other hand, GRN, TMEM106B, ABCC9, and KCNMB2 have been reported to harbor risk alleles associated with HS-Aging pathology. Although GWAS have succeeded in revealing numerous susceptibility variants for dementias, it is an ongoing challenge to identify functional loci and to understand how they contribute to dementia pathogenesis. Until recently, rare variants were not investigated comprehensively. GWAS rely on genotype imputation which is not reliable for rare variants. Therefore, imputed rare variants are typically removed from GWAS analysis. Recent advances in sequencing technologies enable accurate genotyping of rare variants, thus potentially improving our understanding the role of rare variants on disease. There are significant computational and statistical challenges for these sequencing studies. Traditional single variant-based association tests are underpowered to detect rare variant associations. Instead, more powerful and computationally efficient approaches for aggregating the effects of rare variants have become a standard approach for association testing. The sequence-kernel association test (SKAT) is one of the most powerful rare variant analysis methods. A recently-proposed scan-statistic-based test is another approach to detect the location of rare variant clusters influencing disease. In the first study, we examined the gene-based associations of the four putative risk genes, GRN, TMEM106B, ABCC9, and KCNMB2 with HS-aging pathology. We analyzed haplotype associations of a targeted ABCC9 region with HS-Aging pathology and with ABCC9 gene expression. In the second study, we elucidated the role of the non-coding SNPs identified in the International Genomics of Alzheimer’s Project (IGAP) consortium GWAS within a systems genetics framework to understand the flow of biological information underlying AD. In the last study, we identified genetic regions which contain rare variants associated with AD using a scan-statistic-based approach.

Abstract Older adults with complex memory problems benefit when they have the opportunity to consult with a specialist for a comprehensive cognitive assessment. Specialists such as geriatricians, psychogeriatricians or neurologists often work in major cities or large metropolitan towns. Travelling to see a specialist either alone or with a carer is complicated for an older person because of medical issues or disability. The use of videoconferencing may provide a way to link a specialist with a patient without the need for the patient, or the specialist, to travel long distances. Two literature reviews were carried out. The initial review identified studies of the diagnosis of cognition via telemedicine. Thirty-two studies were identified which assessed cognition via telephone or video conference. The focus of the study was either the administration of a standardised cognitive assessment tool (n=30) or an unstructured comprehensive cognitive assessment interview (all via video conference) (n=2). The sample sizes were small but the levels of agreement were high, suggesting that further work in this area may identify that diagnosis of dementia via video conference is reliable. There has been limited work in the area of diagnostic agreement when a specialist is assessing a patient for the first time via video conference, even less work in the area of mental health assessment of older people. The second literature review identified 19 studies of diagnostic agreement using video conference with a sample size of 20 or more. The fields of research were: Dermatology (n=10); Mental Health (n=4); Minor Injuries (n=2); Neurology (n=2); and Rheumatology (n=1). Of the four studies in the area of mental health, one focused on the diagnosis of dementia with the publication of a protocol for assessing Alzheimer’s disease (AD) via video conference. The review highlighted that diagnosis via video conference in other medical fields had been shown to be reliable but that limited work was evident regarding the reliability of diagnosing dementia via video conference. A range of statistical analyses have been used to measure agreement in studies of diagnosis via VC. Overall Proportional Agreement (Po) and Cohen’s kappa (K) are the two most common calculations. There was little uniformity of reporting in the studies identified in the literature review. The variation in reporting made it difficult to compare results or provide data for a meta-analysis of similar studies. Consideration of the methods for analysing diagnostic agreement was undertaken using the approaches identified in the literature review as a starting point. The aim of this research was to identify if a diagnosis of the presence of dementia in an older adult by a specialist is reliable when the assessment interview occurs via video conference. A pilot study was carried out prior to the implementation of a National Health and Medical Research Council (NHMRC) funded multi-site project to test inter-rater agreement for the diagnosis of dementia and subsidiary questions. The candidate is a Chief Investigator (CI) on the NHMRC project grant and participated in writing the grant submission. A pilot study was completed for the purpose of refining the research protocol and establishing preliminary data for the calculation of sample size. A geriatrician carried out a cognitive assessment via video conference with the patient and the carer. The doctor had access to the patient’s chart and the results of a battery of standardised cognitive assessments administered face-to-face (FTF) by the clinic nurse earlier in the day. A second interview was carried out, face-to-face, by a second doctor on the same day. Inter-rater reliability was assessed between doctors. To place the level of agreement in context, inter-rater reliability between paired face-to-face assessments was also measured. Forty-two participants were divided into two groups: paired face-to-face assessments (FF, n=22) or paired video conference and face-to-face assessments (FV/VF, n=20). Twenty-two participants were male. Their average age was 70 years (SD=11.1, Range 50-90). The mean Standardised Mini-Mental State Examination (SMMSE) score was 23.93 (SD=5.42, Range 8-30)[1]. The outcome of agreement was measured using PO and Cohen’s K. FF group (PO=0.636; K=0.430, p=0.005) and the FV/VF group (PO=0.650; K=0.650, p=0.004) showed similar levels of agreement. The main study was a non-inferiority, prospective cohort study following a similar format as the pilot. Patients were randomised to two groups. The first group participated in two interviews; one via VC, the other was face-to-face (FV/VF). The second group was subjected to dual face-to-face assessments (FF). The video conference interview consisted of real-time video conference with the doctor interacting with both the carer and the patient. A battery of standardised assessments, a medical history, imaging and blood tests were prepared before hand and available to the doctor for use in the interview. One hundred and fifty-five participants were divided into two groups: paired face-to-face assessments (FF, n=73) or paired video conference and face-to-face assessments (FV or VF, n=82). Seventy-five of the participants were male. The average age was 76-years (SD=9, Range 54-95). The mean SMMSE was 23.8 (SD=4.4, Range 8-30). Overall proportional agreement (Po) and Weighted K were calculated as a measure of agreement for the presence of dementia. The FF group (Po=0.740; K=0.57, p<0.0001) and the FV/VF group (Po=0.780; K=0.64, p<0.0001) showed similar levels of agreement. Using the same study cohort, additional data were collected to identify the significance of the physical examination (PE) for diagnosing dementia. One of two doctors was allocated the task of completing a physical examination of the patient following initial assessment interview and after the diagnosis decisions had been recorded. Following the physical examination a second record of diagnosis decisions were recorded by the same doctor. The doctor was asked to identify if the diagnosis, formulation, treating options or additional investigations had altered as a result of the in-person physical examination. The physical examination supported clarity of the diagnosis particularly if a vascular element was involved. This dissertation provides evidence of the reliability of a diagnosis of dementia obtained via video conference. Furthermore, the work detailed in this dissertation represents the largest international study on assessing diagnostic accuracy of cognition and makes a significant contribution to the work in telemedicine in the area of mental health. This dissertation provides generalisations that can impact the use of video conference for diagnosis across a range of specialities, with the ultimate goal of improving access to specialist advice for people living in rural areas.