This thesis examines the utility of USPIO-MRI to provide a tool of tracking macrophage recruitment to sites of neuroinflammation within the CNS. Recruited macrophages and microglia resident in CNS tissue play a key role in the pathophysiology of a number of neuroinflammatory diseases such as neuropathic pain and multiple sclerosis. Under activated conditions, microglia and macrophages will phagocytose invading cells and CNS debris. It has been shown that ultrasmall superparamagnetic particles of iron oxide (USPIO), such as Sinerem, injected systemically, are engulfed by macrophages, which in turn migrate to sites of tissue injury. USPIOs can be visualised as a distinct reduction in signal intensity on T2* weighted MR images. However, there are still some issues regarding the distinction between iron-laden recruited macrophages and the entry of free iron across a permeable blood brain barrier (BBB) in disease cases. Hence, it was shown that intravenously injected Sinerem is cleared from the peripheral circulation within 24 hours, indentifying this as a time point as suitable for MCP-1 injection. Data showed that free USPIO can be visualised in the brain and that there is a linear relationship between Sinerem concentration and T2* signal intensity changes. MCP-1 induces macrophage recruitment to the site of microinjection and causes BBB breakdown at between 3 and 4 hours. In particular it was shown that T2* signal intensity changes are seen, in the presence of an intact BBB, as a result of Sinerem laden macrophages. This finding was verified by the co-localisation of ED-1 positive cells and Prussian blue positive regions. It was demonstrated that there is a strong correlation between T2* signal changes and the number of macrophages. This demonstrates that USPIO-MRI can be used to characterise macrophage infiltration in neuroinflammation in the presence of an intact BBB.

O objetivo deste estudo foi verificar o comportamento anatomopatológico e expressão imunohistoquímica das proteínas GFAP, Vimentina, COX-2 e Amilóide β em 14 casos de encefalopatias não infecciosas em cães, mais especificamente Meningoencefalite Granulomatosa (MEG), Meningoencefalite Necrotizante (MEN) e Angiopatia Amilóide Cerebral (CAA). Foram coletadas informações clínicas (gênero, raça, idade) e morfológicas (necrose, presença de proteína amilóide, infiltrado inflamatório) dos animais. Para a expressão das proteínas por imunohistoquímica foram confeccionadas lâminas próprias para tal com amostras dos tecidos, juntamente com controles positivos das reações. A avaliação da expressão das proteínas foi de como positivo ou negativo para a marcação para GFAP, Vimentina e COX-2, e para o Aβ seguiu-se a classificação proposta por Olichney (1995), com quatro graduações. Animais SRD (4/14, 28%), de raça Maltês (2/14, 14%), Labrador (2/14, 14%), Poodle (3/14, 21%), Fox Terrier (1/14, 7%), Pug (1/14, 7%) e Bichon Frisè (1/14, 7%) fizeram parte deste estudo, na maioria machos (9/14, 63%). As lesões em todos os casos foram características, sendo que em MEG foi observado manguitos perivasculares abundantes com infiltrado inflamatório disperso pelo parênquima encefálico, caracterizando os quatro casos como de MEG disseminada. Em MEN foi observado o infiltrado inflamatório acompanhado de áreas de necrose, sinais característicos da doença. Nos casos de CAA foi observado agregados proteicos junto aos vasos sanguíneos do encéfalo, confirmados como sendo material amilóide por meio da coloração Vermelho Congo. Quanto a expressão dos antígenos por imunohistoquímica, houve a marcação de todos anticorpos em todos os casos, e, para os casos de CAA, houve uma maioria de casos com classificação 1 (5/6, 83%). Em conclusão: 1. os dados clínicos obtidos reproduzem o comportamento biológico destas doenças em cães; 2. a análise imunohistoquímica das lesões de MEG, MEN e CAA apresentou resultados dentro dos quadros descritos na literatura; 3. a marcação positiva para COX-2 indica um aumento na atividade macrofágica no encéfalo; 4. as marcações de GFAP e Vimentina indicam uma reação das células da glia frente a lesão apresentada; 5. a utilização da imunohistoquímica como ferramenta de diagnóstico para estas doenças é válida. / The goal of this study was to analyze the anatomopathological and immunohistochemical profile for GFAP, Vimentin, COX-2 and β-amyloid in 14 cases of noninfectious encephalopathies in dogs, more specifically Granulomatous Meningoencephalitis (GME), Necrotizing Meningoencephalitis (NME) and Cerebral Amyloid Angiopathy (CAA). Clinical (gender, breed, age) and morphological (necrosis, presence of amyloid protein, inflammatory infiltrate) informations were collected regarding such animals. Microscopy slides with paraffin-embedded sections of tissue were elaborated for the immunohistochemical analysis, as well as positive controls for the reactions. The expressions of the proteins were graded as positive or negative for GFAP, Vimentin and COX-2, and the grading system described by Olichney was used for the β-amyloid. Animals of mixed breed (4/14, 28%), malteses (2/14, 14%), Labrador (2/14, 14%), Poodle (3/14, 21%), Fox Terrier (1/14, 7%), Pug (1/14, 7%) and Bichon Frisè (1/14, 7%) featured in this study, and most of them were males (9/14, 63%). All of the lesions observed were characteristic of such diseases, as perivascular cuffing with an abundant inflammatory infiltrate was observed in GME cases, being all 4 cases considered as disseminated GME. Necrosis, as well as the inflammatory infiltrate, was observed in all NME cases, which is the hallmark of this disease. The deposition of amyloid protein was observed in cases of CAA, which were confirmed by the Congo Red special staining. As for the immunohistochemical expression, all antibodies performed positive staining, and for the β-amyloid there was a predominance of grade 1 cases (5/6, 83%). In conclusion: 1. the clinical data reproduce the biological behavior of such diseases in dogs; 2. the immunohistochemical analysis of the lesions from GME, NME and CAA presented results coherent with those found in the literature; 3. positive staining for COX-2 presented an increased macrophage activity in the brain of those animals; 4. GFAP and Vimentin positive staining indicate a reaction by the glial cells against the presented lesion; 5. immunohistochemistry is a valid diagnostics tool for such diseases.

O objetivo deste estudo foi verificar o comportamento anatomopatológico e expressão imunohistoquímica das proteínas GFAP, Vimentina, COX-2 e Amilóide β em 14 casos de encefalopatias não infecciosas em cães, mais especificamente Meningoencefalite Granulomatosa (MEG), Meningoencefalite Necrotizante (MEN) e Angiopatia Amilóide Cerebral (CAA). Foram coletadas informações clínicas (gênero, raça, idade) e morfológicas (necrose, presença de proteína amilóide, infiltrado inflamatório) dos animais. Para a expressão das proteínas por imunohistoquímica foram confeccionadas lâminas próprias para tal com amostras dos tecidos, juntamente com controles positivos das reações. A avaliação da expressão das proteínas foi de como positivo ou negativo para a marcação para GFAP, Vimentina e COX-2, e para o Aβ seguiu-se a classificação proposta por Olichney (1995), com quatro graduações. Animais SRD (4/14, 28%), de raça Maltês (2/14, 14%), Labrador (2/14, 14%), Poodle (3/14, 21%), Fox Terrier (1/14, 7%), Pug (1/14, 7%) e Bichon Frisè (1/14, 7%) fizeram parte deste estudo, na maioria machos (9/14, 63%). As lesões em todos os casos foram características, sendo que em MEG foi observado manguitos perivasculares abundantes com infiltrado inflamatório disperso pelo parênquima encefálico, caracterizando os quatro casos como de MEG disseminada. Em MEN foi observado o infiltrado inflamatório acompanhado de áreas de necrose, sinais característicos da doença. Nos casos de CAA foi observado agregados proteicos junto aos vasos sanguíneos do encéfalo, confirmados como sendo material amilóide por meio da coloração Vermelho Congo. Quanto a expressão dos antígenos por imunohistoquímica, houve a marcação de todos anticorpos em todos os casos, e, para os casos de CAA, houve uma maioria de casos com classificação 1 (5/6, 83%). Em conclusão: 1. os dados clínicos obtidos reproduzem o comportamento biológico destas doenças em cães; 2. a análise imunohistoquímica das lesões de MEG, MEN e CAA apresentou resultados dentro dos quadros descritos na literatura; 3. a marcação positiva para COX-2 indica um aumento na atividade macrofágica no encéfalo; 4. as marcações de GFAP e Vimentina indicam uma reação das células da glia frente a lesão apresentada; 5. a utilização da imunohistoquímica como ferramenta de diagnóstico para estas doenças é válida. / The goal of this study was to analyze the anatomopathological and immunohistochemical profile for GFAP, Vimentin, COX-2 and β-amyloid in 14 cases of noninfectious encephalopathies in dogs, more specifically Granulomatous Meningoencephalitis (GME), Necrotizing Meningoencephalitis (NME) and Cerebral Amyloid Angiopathy (CAA). Clinical (gender, breed, age) and morphological (necrosis, presence of amyloid protein, inflammatory infiltrate) informations were collected regarding such animals. Microscopy slides with paraffin-embedded sections of tissue were elaborated for the immunohistochemical analysis, as well as positive controls for the reactions. The expressions of the proteins were graded as positive or negative for GFAP, Vimentin and COX-2, and the grading system described by Olichney was used for the β-amyloid. Animals of mixed breed (4/14, 28%), malteses (2/14, 14%), Labrador (2/14, 14%), Poodle (3/14, 21%), Fox Terrier (1/14, 7%), Pug (1/14, 7%) and Bichon Frisè (1/14, 7%) featured in this study, and most of them were males (9/14, 63%). All of the lesions observed were characteristic of such diseases, as perivascular cuffing with an abundant inflammatory infiltrate was observed in GME cases, being all 4 cases considered as disseminated GME. Necrosis, as well as the inflammatory infiltrate, was observed in all NME cases, which is the hallmark of this disease. The deposition of amyloid protein was observed in cases of CAA, which were confirmed by the Congo Red special staining. As for the immunohistochemical expression, all antibodies performed positive staining, and for the β-amyloid there was a predominance of grade 1 cases (5/6, 83%). In conclusion: 1. the clinical data reproduce the biological behavior of such diseases in dogs; 2. the immunohistochemical analysis of the lesions from GME, NME and CAA presented results coherent with those found in the literature; 3. positive staining for COX-2 presented an increased macrophage activity in the brain of those animals; 4. GFAP and Vimentin positive staining indicate a reaction by the glial cells against the presented lesion; 5. immunohistochemistry is a valid diagnostics tool for such diseases.

The main objective of this study was to elucidate the possible mechanistic link between traumatic brain injury (TBI) and Alzheimer's disease (AD) using an animal model. We examined behavioral and histological effects of TBI in pre-symptomatic AD-transgenic mice (C57B6/SJL/SwissWebster/B6D2F1). In previous studies, these mice displayed AD-like behavioral deficits by 15-17 months of age and AD-like neuropathology as early as six months of age. To clarify the effects of TBI on these mice, the present study began when they were about three months of age and the study ended when they were about five months of age. As a control, non-transgenic (NT) mice were also evaluated in this study. To assess behavioral changes following TBI, all mice were subjected to 14 days of pre-TBI training of a spatial memory task, the radial arm water maze (RAWM). After training, there were no performance differences between AD-transgenic mice and NT mice. Then, half of the AD-transgenic mice, as well as half of the NT mice, received an experimental TBI at the right parietal cortex using a pneumatic impactor. The other half of these mice received sham surgery. At two, four, and six weeks after surgery, all mice were tested in the same water maze task and the numbers of errors were recorded. AD-transgenic mice with TBI made significantly more errors than AD-transgenic mice without TBI and NT mice regardless of TBI. Furthermore, deficits were observed at both two and six weeks after TBI surgery. To assess histological changes following TBI, we used a monoclonal antibody against beta-amyloid to detect AD-like plaques and an antibody against NeuN to evaluate the total neuronal loss. There were no clear group differences in terms of the beta amyloid expression pattern, although one AD-transgenic mouse with TBI showed AD-like beta amyloid plaques throughout the entire cortex and hippocampus. These results suggest that TBI precipitated behavioral deficits in a spatial memory task in pre-symptomatic AD-transgenic mice, but not control mice. Further studies are warranted for histological effects of TBI.

Le contact par le regard est connu pour générer divers effets sur la cognition et le comportement humain. Il améliore notamment l’encodage des visages, favorise la sélection de comportements prosociaux et influence positivement le jugement que nous formons sur autrui. Cette thèse a pour objectifs de mieux comprendre les mécanismes sous-tendant ces effets et d’investiguer leur potentiel thérapeutique. La revue exhaustive des effets du contact par le regard sur la cognition humaine, nous a amené à considérer ces effets sous un nouveau jour. Selon cette perceptive, le contact par le regard induirait des processus de référence à soi, une fonction mentale qui consiste à assigner une valeur personnelle aux objets et aux événements rencontrés. Dans la lignée de cette hypothèse, cette thèse démontre tout d’abord que le contact par le regard améliore la conscience intéroceptive (Expérience 1). Des résultats obtenus à l’aide de l’imagerie cérébrale (Expérience 2) suggèrent fortement que cet effet est effectivement sous-tendu par des mécanismes d’autoréférencement. Par ailleurs, cette thèse met pour la première fois en valeur que les effets du contact par le regard sont en majorité bénéfiques et qu’ils pourraient donc bénéficier à certaines populations souffrant d'anomalies précisément dans les mécanismes influencés par le contact par le regard. Dans la lignée de cette hypothèse, nous démontrons que les effets du contact par le regard sur la mémoire des visages et les évaluations d’autrui sont présents chez les patients atteints de maladie d’Alzheimer (Expérience 3) suggérant que ces effets pourraient être utilisés pour améliorer la qualité de leurs interactions sociales. / Eye contact is known to have diverse effects on human cognition and behaviour. It stimulates memory for faces, promotes prosocial actions and increases positive appraisal of others. This thesis aims at better characterizing the mechanisms underlying the eye contact effects and investigating their therapeutic potential. The exhaustive review of the eye contact effects leads us to the proposal that these effects are by-products of self-referential processing evoked by eye contact, i.e. a cognitive mechanism designed to attribute personal meaning to stimuli. In line with this view, we demonstrated that eye contact enhances interoceptive self-awareness (Experiment 1). Results obtained using brain imagery (Experiment 2) strongly suggest that this effect is indeed subtended by self-referential processing. Moreover, we explicitly emphasize that the eye contact effects reflect positive impacts on human cognition, and as such may open new ways of improving the quality of life in individuals with various conditions where the mechanisms improved by eye contact are precisely impaired. In line with this proposal, we demonstrated that the effects of eye contact on memory for faces and on other’s appraisal are preserved in patients with Alzheimer’s Disease (Experiment 3). Therefore, these effects may be used to enhance the quality of their social exchanges.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In the first portion of this thesis, epidemiology, clinical signs and distribution of lesions in the brain of thirty one cases of polioencephalomalacia (PEM) in cattle from the Southern (13 cases) and Midwestern (18 cases) Brazil were studied. Morbidity (0.04%-6.66 %), mortality (0.04%-6.66 %), and lethality (50%-100%) rates were similar in both regions studied. Cases occurred mainly in cattle raised at pasture; in the Southern the disease affected mainly young cattle while mainly older cattle were affected in the Midwest. Clinical signs more frequently observed included blindness, incoordination, circling, opisthotonus, recumbence and paddling movements. Clinical course varied from 12 hours to 8 days. In 11 cases no gross changes were observed in the brain. Main gross findings in the brain of remaining cases included congestion with swelling and flattening of gyri, softening and yellow discoloration of cerebral cortex, hemorrhagic foci in the brain stem, cerebellum and telencephalon, and cerebellar herniation. The main histopathological changes were in the cortex of occipital, parietal and frontal telencephalic lobes; however less prominent and less frequently found lesions occurred in the hippocampus, basal nuclei, thalamus, midbrain, and cerebellum. The type of microscopic cortical lesions was consistent in all cases and included segmentar laminar neuronal necrosis, spongiosis, swollen of vascular endothelial nuclei, Alzheimer type II astrocytes and infiltration of gitter cells. In 20% of the cases there was mild lymphohistiocytic cellular infiltrate and in 13% of the cases there was mild infiltrate by neutrophils and eosinophils. Additionally, mild to moderate necro-hemorrhagic lesions were observed in 49% of the cases in the basal nuclei, in 39% of the cases in brain stem and in 26% of the cases in the thalamus. In the cortical laminae of the occipital, parietal and frontal telencephalic lobes, most frequently affected cortical layers both by neuronal necrosis and edema were external and internal granular layers. Both gyri and sulci were equally affected. In the second portion of the thesis, one experimental model for the study of the etiology, pathology, and pathogenesis of polioencephalomalacia in ruminants was established. The condition was induced in five sheep by oral administration of amprolium at daily doses of 500 and 1,000 mg per Kg of live body weight respectively for 28-59 days and for 13-39 days. Clinical course varied from 3 to 7 days. Clinical signs included depression, incoordination, midriasis, grinding of the teeth, blindness, and laying down with opisthotonus and paddling movements. Drooling and a sawhorse stance were observed in one sheep and myoclonus in another one. Main gross lesions were restricted to the central nervous system and included swelling of the brain with flattening of telencephalic gyri, and hemorrhages in the parietal and occipital lobes of the telencephalon. Histologically, there was segmental laminar neuronal necrosis (red neurons) associated with edema, swelling of endothelial cells, hemorrhages and infiltration by foamy macrophages (gitter cells). These changes were more marked in the frontal, parietal and occipital telecephalic lobes and there was sharp demarcation between the lesions and the adjacent normal neuropile. Additionally, similar, but less marked lesions were seen in the mesencephalon, thalamus and hippocampus. Considering the consistent reproducible aspects of polioencephalomalacia in sheep using amprolium, this may be an useful model for the study of the disease. / Na primeira parte dessa tese, foram estudados a epidemiologia, os sinais clínicos e a distribuição das lesões no encéfalo de 31 casos de polioencefalomalacia (PEM) em bovinos do Estado do Rio Grande do Sul (13 casos) e da Região Centro-Oeste do Brasil (18 casos). As taxas de morbidade (0.04%-6.66 %), mortalidade (0.04%-6.66 %) e letalidade (50%-100%) foram semelhantes em ambas as regiões estudadas. Os casos ocorreram principalmente em bovinos criados de forma extensiva em pastagem. Na Região Sul a doença afetou principalmente bovinos jovens, enquanto que principalmente bovinos mais velhos foram afetados no Centro-Oeste. Os sinais clínicos mais freqüentemente observados incluíram cegueira, incoordenação, andar em círculos, opistótono, decúbito e movimentos de pedalagem. A evolução do quadro clínico variou de 12 horas a 8 dias. Em 11 encéfalos não foram observadas alterações macroscópicas; as principais alterações macroscópicas nos outros casos incluíam congestão com tumefação e achatamento das circunvoluções, amolecimento e amarelamento do córtex telencefálico, focos de hemorragia no tronco encefálico, cerebelo e telencéfalo e herniação cerebelar. As principais alterações histológicas ocorreram no córtex dos lobos telencefálicos occipital, parietal e frontal; no entanto, lesões menos acentuadas e menos frequentemente observadas ocorreram no hipocampo, núcleos da base, tálamo, mesencéfalo e cerebelo. O tipo de lesão microscópica cortical era consistente em todos os casos e incluía necrose neuronal laminar segmentar, espongiose, tumefação do núcleo das células endoteliais, astrócitos Alzheimer do tipo II e infiltração por células gitter. Em 20% dos casos havia um leve infiltrado celular linfo-histiocitário e em 13% dos casos havia leve infiltrado de neutrófilos e eosinófilos. Adicionalmente, lesões necro-hemorrágicas leves ou moderadas foram observadas em 49% dos casos nos núcleos da base, em 39% dos casos no tronco encefálico e em 26% dos casos no tálamo. Na substância cinzenta dos telencéfalos frontal, parietal e occipital, as camadas granular externa e interna foram as mais afetadas tanto por neurônios necróticos quanto por edema. Tanto os giros quanto os sulcos foram afetados igualmente. Na segunda parte da tese, foi estabelecido um modelo experimental para o estudo da etiologia, patologia e patogênese da polioencefalomalacia em ruminantes. A condição foi induzida em cinco ovinos pela administração oral de amprólio nas doses diárias de 500 e 1.000 mg/Kg de peso vivo, respectivamente por 28-59 dias e 13-39 dias. O curso clínico foi de 3-7 dias. Os sinais clínicos incluíam depressão, incoordenação, midríase, bruxismo, cegueira e decúbito com opistótono e movimentos de pedalagem. Salivação excessiva e posição de cavalete foi observada em um ovino e mioclonias em um outro. Os principais achados de necropsia restringiam-se ao sistema nervoso central e incluíam tumefação do encéfalo com achatamento dos giros telencefálicos e hemorragias nos lobos parietal e occipital do telencéfalo. Histologicamente, havia necrose segmentar laminar de neurônios associada a edema, tumefação de células endoteliais, hemorragias e infiltração por macrófagos espumosos (células gitter). Essas alterações eram mais marcadas nos lobos frontal, parietal e occipital do telencéfalo e havia uma demarcação abrupta entre as lesões e o neurópilo normal adjacente. Adicionalmente, lesões semelhantes, mas menos acentuadas, eram observadas no mesencéfalo, tálamo e hipocampo. Levando em consideração a reproducibilidade regular dos aspectos da polioencefalomalacia em ovinos pela administração de amprólio, esse modelo pode ser útil para o estudo da doença.

Der Morbus Parkinson bzw. das idiopathische Parkinson-Syndrom ist pathophysiologisch durch eine progressive Degeneration der dopaminergen Neurone in der Substantia nigra und im Locus coeruleus charakterisiert. Obwohl die genaue Ursache der Erkrankung bis heute unbekannt ist, wird eine multifaktorielle Genese bei polygenetischer Prädisposition angenommen. Des Weiteren liefert die sogenannte Braak-Hypothese eine mögliche Kausalkette. Diese Hypothese besagt, dass unbekannte Erreger die Fehlfaltung von physiologischem α-Synuclein im peripheren Nervensystem, insbesondere auch im enterischen Nervensystem, initiieren können, welche sich dann retrograd durch axonalen Transport über den Nervus vagus zu dem dorsalen motorischen Kern des Nervus vagus ausbreitet. Von dort werden zusätzliche Hirnregionen, einschließlich der Substantia nigra, beeinträchtigt. Es wird vermutet, dass diese neuronale α-Synuclein-Aggregation im Gehirn ein zentraler Bestandteil der Pathogenese des Morbus Parkinson darstellt. Als histopathologisches Korrelat des Morbus Parkinson können in der Substantia nigra sogenannte Lewy-Körperchen im Zytoplasma der Nervenzellen nachgewiesen werden, die aus intrazellulären Proteinablagerungen und hauptsächlich aus unlöslichem und aggregiertem α-Synuclein bestehen. α-Synuclein-Aggregate im Magen-Darm-Trakt wurden als potenzieller Biomarker für die Früherkennung eines Morbus Parkinson vorgeschlagen. Postuliert wurde hier, dass die α-Synuclein-Pathologie im Darm zeitlich bereits vor der Pathologie in der Substantia nigra nachweisbar ist. Studien, die diese Hypothese weiter untersuchten, führten jedoch zu divergierenden Ergebnissen. Die Zielsetzung der vorgelegten Studie war es, Ablagerungen des α-Synucleins und des phosphorylierten α-Synucleins im menschlichen Gastrointestinaltrakt mithilfe von Immunhistochemie zu untersuchen, um eine Aussage darüber treffen zu können, ob α-Synuclein als potenzieller prädiktiver Biomarker für die Erkrankung geeignet sein könnte. Verwendet wurden hierzu einerseits Antikörper gegen natives α-Synuclein und andererseits Antikörper gegen das an Serin 129 phosphorylierte α-Synuclein, welche die pathologische, aggregierte Form des Proteins besser darstellt. Es sollte festgestellt werden, ob sich die Prävalenz der (phosphorylierten) α-Synuclein-Ablagerungen im Darm von Parkinson-Patienten und Kontrollpatienten unterscheiden. Eine mangelnde Spezifität des Antikörpers gegen das phosphorylierte α-Synuclein wurde dabei durch die Implementierung eines Kontrollexperiments mit einer proteolytischen Vorbehandlung an Gewebeproben histopathologisch bestätigter Parkinson-Patienten ausgeschlossen. In dieser retrospektiven Studie wurde die α-Synuclein-Expression in post-mortem entnommenen Hirn-, Dünn- und Dickdarmproben von 25 neuropathologisch bestätigten Parkinson-Patienten und 20 alters- und geschlechtsspezifisch abgestimmten Kontrollpatienten untersucht. Die Immunoreaktivität wurde durch einen neuen Ansatz quantifiziert, der die detaillierte Bewertung von a-Synuclein-positiven morphologischen Strukturen des enterischen Nervensystems beinhaltet. Alle immungefärbten Schnitte wurden von zwei Untersuchern unabhängig evaluiert. Beide waren bezüglich der Gruppenzuordnung (Gewebeproben von Parkinson-Patienten oder von Kontrollpatienten) verblindet. Zu diesem Zweck wurden von jedem Präparat jeweils 10 zufällig ausgewählte, nicht selektive mikroskopische Bilder/HPF (High-power Fields) in einer 100-fachen Vergrößerung aufgenommen. Insgesamt wurden 1620 HPFs von jedem Untersucher hinsichtlich einer positiven Immunoreaktivität in den drei morphologischen Strukturen „Nervenfasern/Einzelfasern“, „Plexus“ und „Ganglienzellen“ beurteilt. Die Auswertung der Ergebnisse ergab, dass die Immunoreaktivität von α-Synuclein und phosphoryliertem α-Synuclein bei Parkinson-Patienten im Vergleich zu den Kontrollen in fast jeder der untersuchten morphologischen Strukturen signifikant reduziert war. Bis auf die Einzelfasern im Dickdarm bei der immunhistochemischen Färbung mit Antikörper gegen nativem α-Synuclein, sah man durchgehend signifikant weniger Immunoreaktivität in den verschiedenen morphologischen Strukturen im Darmgewebe der Parkinson-Patienten verglichen zu denen der Kontrollpatienten. Der immunhistochemische Nachweis von α-Synuclein und phosphoryliertem α-Synuclein scheint demzufolge ein häufiger und potenziell normaler Befund zu sein. Weder α-Synuclein noch phosphoryliertes α-Synuclein kann daher als molekularer Biomarker der Parkinson-Pathologie betrachtet werden. Die reduzierte intestinale Immunoreaktivität bei Parkinson-Patienten spiegelt eher die Parkinson-bedingte neuronale Degeneration wider. Das Resultat dieser Studie legt nahe, dass der Nachweis von α-Synuclein- und phosphoryliertem α-Synuclein in intestinalen Nervenfasern, dem intestinalen Plexus und intestinalen Ganglienzellen einem Normalbefund entspricht und nicht einer Morbus Parkinson-assoziierten Pathologie. Als bis heute größte Post-mortem-Studie, welche α-Synuclein und phosphoryliertes α-Synuclein in Gewebeproben des Gastrointestinaltraktes von histopathologisch bestätigten Parkinson-Patienten untersuchte, liefert die vorliegende Studie einen wichtigen Beitrag zur Erforschung von α-Synuclein im Gastrointestinaltrakt bei Parkinson-Patienten. Der Nachweis von α-Synuclein in gastrointestinalem Gewebe erscheint für die Diagnostik, insbesondere für die klinische Prädiktion einer Parkinson-Krankheit, ungeeignet.:1. Einführung 1.1. Morbus Parkinson 1.2. Ätiologie und Epidemiologie des idiopathischen Parkinson-Syndroms 1.3. Neuropathologie des idiopathischen Parkinson-Syndroms 1.4. α-Synuclein und phosphoryliertes α-Synuclein 2. Die Bedeutung von (phosphoryliertem) α-Synuclein als molekularer Biomarker bei Morbus Parkinson: aktuelle Studienlage 3. Ableitung der Rationale für die publizierte Studie 3.1. Grundlagen 3.2. Studienziele 3.2.1. Nachweis von α-Synuclein und phosphoryliertem α-Synuclein im Dünndarm und Dickdarm von Parkinson-Patienten und Kontrollpatienten 3.2.2. Vergleich von α-Synuclein und phosphoryliertem α-Synuclein im Dünndarm und Dickdarm von Parkinson-Patienten und Kontrollpatienten 4. Methodik 4.1. Patientenkollektiv 4.2. Gewebeverarbeitung 4.3. Immunhistochemie 4.3.1. α-Synuclein 4.3.2. phosphoryliertes α-Synuclein 4.4. Beurteilung der immunhistochemischen Färbung und Quantifizierung der morphologischen Strukturen 5. Publikationsmanuskript 6. Zusammenfassung der Arbeit 7. Literaturverzeichnis Erklärung über die eigenständige Abfassung der Arbeit Lebenslauf Danksagung

This thesis investigates rest-activity and sleep profiles in neurodegeneration and brain cancer. Study 1 comprised longitudinal field assessments of rest-activity, sleep and memory in controls and memory-impairment individuals with: subjective memory complaint (SMC), amnestic mild cognitive impairment (aMCI), mild and moderate Alzheimer’s disease (AD). Four questions were addressed: (1) is SMC a prodromal stage of AD? (2) do characteristics of SMC predict future decline? (3) does cholinergic medication (ChEI) impact rest-activity and sleep of moderate AD patients? and (4) are there factors predicting response to ChEI? Study 2 assessed rest-activity and melatonin rhythms in a brain cancer patient (JJB), and post-mortem analysis of brain tissue assessed infiltration of cancer cells on the circadian clock (SCN). Both studies used questionnaires, cognitive tests, electroencephalography and actigraphy simultaneously at patients’ homes. In Study 1, the SMC group showed a reduced activity amplitude to be correlated with increasing memory impairment severity, lower sleep quality and efficiency. Increased sleep fragmentation was observed in all memory-impaired groups, although not correlated to impairment severity. Increased fragmentation of rest-activity rhythm correlated with increasing memory impairment severity in all groups except SMC. Following ChEI medication with donepezil, moderate AD patients showed increased sleep fragmentation, probably due to potentiation of available acetylcholine known to maintain arousal. Higher daytime-activity and lower activity in the rest-phase, when drug-naïve, predicted improved cognition following ChEIs. In Study 2, cancer cell infiltration of the patient’s SCN was confirmed. However, a robust circadian rest-activity period with a misaligned melatonin phase, was recorded, indicating that the effects of partial SCN lesions in humans are complex and this result was possibly in part are due to the masking effect of social behaviour.

Despite the association between hippocampal atrophy and a vast array of highly debilitating neurological diseases, such as Alzheimer’s disease and frontotemporal lobar degeneration, tools to accurately and robustly quantify the degeneration of this structure still largely elude us. In this thesis, we firstly evaluate previously-developed hippocampal segmentation methods (FMRIB’s Integrated Registration and Segmentation Tool (FIRST), Freesurfer (FS), and three versions of a Classifier Fusion (CF) technique) on two clinical MR datasets, to gain a better understanding of the modes of success and failure of these techniques, and to use this acquired knowledge for subsequent method improvement (e.g., FIRSTv3). Secondly, a fully automated, novel hippocampal segmentation method is developed, termed Fast Marching for Automated Segmentation of the Hippocampus (FMASH). This combined region-growing and atlas-based approach uses a 3D Sethian Fast Marching (FM) technique to propagate a hippocampal region from an automatically-defined seed point in the MR image. Region growth is dictated by both subject-specific intensity features and a probabilistic shape prior (or atlas). Following method development, FMASH is thoroughly validated on an independent clinical dataset from the Alzheimer’s Disease Neuroimaging Initiative (ADNI), with an investigation of the dependency of such atlas-based approaches on their prior information. In response to our findings, we subsequently present a novel label-warping approach to effectively account for the detrimental effects of using cross-dataset priors in atlas-based segmentation. Finally, a clinical application of MR hippocampal segmentation is presented, with a combined MR-PET analysis of wholefield and subfield hippocampal changes in Alzheimer’s disease and frontotemporal lobar degeneration. This thesis therefore contributes both novel computational tools and valuable knowledge for further neurological investigations in both the academic and the clinical field.

I have sought to discover genetic causes of neuroanatomical defects by conducting N-ethyl-N-nitrosourea mutagenesis and transgenic knock out screens in mice. The rationale behind this is that mutations causal to structural defects will be informative about developmental neurobiology and the biological basis of behaviour. Direct screening for behavioural abnormalities in mice has historically been arduous and yielded few findings due to small effect sizes and limited statistical power. My approach sought to bypass these problems by screening for highly penetrant morphological phenotypes. This thesis details my screens and the histological, genetic and behavioural characterisation of lines of interest. These include models of hydrocephalous, pyramidal cell layer ectopia, abnormal neurogenesis, corpus callosum agenesis, hippocampal enlargement, elevated cell death and hypomyelination. Whilst N-ethyl-N-nitrosourea mutagenesis screening has been conducted since the twilight of the 20^th century, systematic transgenic knock out screening is currently in its infancy. By discovering gene-phenotype associations through both approaches, I have been able to compare the relative yields, strengths and weaknesses of the two screening methods. Additionally, I have discussed the significant of the gene-phenotype associations produced from both screens.