Tesi sul tema "Olfactory disorders"

Olfactory identification deficits have received recent attention as a potentially useful endophenotype for schizophrenia. Examination of this deficit in individuals with schizotypal personality features (SPF) offers an alternative approach to multiple confounds present when examining individuals with schizophrenia. The aim of the current study was to compare the relative sensitivity of performance on measures of olfaction identification and sustained attention to the presence of SPF. Twenty-six undergraduates were defined as having SPF based on scoring in the top 10% of the Abbreviated Schizotypal Personality Questionnaire (SPQ-B; mean age 19.6, SD = 1.1; 62% female). These individuals were compared to twenty-six controls (scoring lower than half a standard deviation above the mean; mean age 19.8, SD = 1.6; 62% female). All participants were administered the Schizotypal Personality Disorder (SPD) section of the Structured Clinical Interview for DSM IV Axis II Personality Disorders (SCID-II). In addition, participants were administered the Brief Smell Identification Test (B-SIT) and a six-minute degraded-stimuli Continuous Performance Test (CPT). Group differences in performance indices of the CPT did not approach statistical significance. Similarly, there were no statistically significant group differences for males or females in performance on the B-SIT. Correlational analyses examined cognitive performance with a dimension score derived by summing quantitative ratings from the SPD items on the SCID-II. The SPD dimension score showed a statistically significant positive correlation with several performance indices of the CPT, including omission errors (rs(52) = .51, p < .001) and commission errors (rs(52) = .38, p < .005). In contrast, the B-SIT scores were not correlated with the SPD dimension score for males or females. Contrary to our hypothesis, results from the current study suggest that olfactory identification deficits may not represent a robust endophenotype consistently found in samples with schizotypal personality features. With regard to sustained attention, our differential findings suggest that schizotypal traits may be more adequately assessed through an interview by trained clinicians who use clinical judgment to determine the presence of phenotypic aspects of SPD (e.g., SCID-II), rather than relying on self-report measures (e.g., SPQ-B). Implications as well as limitations and future directions of these findings are discussed.
M.S.
Department of Psychology
Sciences
Psychology PhD

Parkinson’s disease (PD) is a complex neurodegenerative disorder influenced by a combination of genetic and environmental factors. The molecular mechanisms that underlie PD are unknown. However, oxidative stress and impairment of antioxidant defence mechanisms have been implicated as major contributors to disease pathogenesis. Previously, we have reported a PD patient-derived cellular model, generated from biopsies of the olfactory mucosa, termed hONS cells. These cells have demonstrated disease-specific differences in gene expression and metabolic activity associated with the Nrf2-mediated antioxidant defence pathway. To date, few studies have examined the role of the Nrf2 encoding gene, NFE2L2, in PD. This thesis comprehensibly assessed whether rare and common NFE2L2 genetic variations modify susceptibility to PD using a large Australian case-control sample (PD=1,338; controls=1,379). We employed a haplotype-tagging approach that identified an association with the tagging SNP rs2364725 and PD (OR = 0.849 (0.760-0.948), P = 0.004). Further genetic screening for rare variants in patient-derived cell lines produced no obvious pathogenic variants in the coding regions of NFE2L2. In addition, we were able to identify some age-at-onset modifying SNPs and replicate an ‘early-onset’ haplotype that contains a previously identified ‘functional promoter’ SNP (rs6721961).
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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Adult neurogenesis is a dynamic field of study, with potential for generating therapies for neurodegenerative disorders. The demonstration of adult brain neurogenesis strengthens the rationale for regenerative therapies for neurodegenerative disorders. The ‘ventriculo-olfactory neurogenic system’ (VONS) is used in the study neurogenesis, migration and differentiation of neural stem cells. Importantly, subventricular zone (SVZ) neuroblasts recapitulate a population of dopaminergic neurons within the olfactory bulb (OB), possibly the only population of dopaminergic interneurons, generated throughout adult life. This study aims to simulate a patho physiological model for olfactory epithelial cell turnover to demonstrate a link between the olfactory epithelium (OE) and cell proliferation in the SVZ. In order to examine the relationship between the OE and the SVZ, an ablation protocol was generated. Using a double dose of methimazole, the OE was completely ablated leading to denervation of the olfactory bulb. This denervation resulted in an increase of astrocyte and microglial activity on day 11 which remained elevated up to day 21. This was accompanied by a significant decrease of tyrosine hydroxylase immunoreactivity in type I periglomerular cells on day 17. While there was a decrease in the type II periglomerular cells on day 17, this was shown to be not significant.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
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BACKGROUND: The present Doctoral Thesis is focused on Rapid-eye-movement (REM) sleep behavior disorder (RBD) and olfactory dysfunction as biomarkers of Synucleinopathies, such as Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). RBD is a parasomnia characterized by loss of atonia during REM sleep that provokes dream-enacting behaviors. Clinically isolated RBD (IRBD) is considered a prodromal stage of alpha-Synucleinopathies since almost 75% of patients after 12 years of the disease develop a PD or atypical parkinsonism. Specifically, in a multicentric study, 56.5% of IRBD patients converted to parkinsonism as the first manifestation, while 43.5% developed dementia first. Subtle motor symptoms, hyposmia, and cognitive impairment are the best predictors of phenoconversion in IRBD. OBJECTIVES AND HYPOTHESES: The main objectives of this Thesis are: 1) To characterize structural and functional brain substrates underlying Idiopathic REM sleep behavior disorder as well as to relate these findings to cognitive performance; 2) To study olfactory dysfunction as a preclinical and clinical biomarker of alpha- Synucleinopathies, and its progression throughout the disease; 3) To investigate progressive brain degeneration throughout IRBD, and to investigate how these changes relate to cognitive decline. The main hypotheses are: 1) IRBD will be characterized by specific changes in brain structure and functional connectivity that will be associate with olfactory and cognitive impairment, 2) IRBD patients will show structural brain changes over time and a cognitive decline superior to that seen in normal aging, 3) Smell dysfunction will be identified in IRBD and PD, and it is expected to progress slightly with the disease course. MATERIAL AND METHODS: This Doctoral Thesis is presented as a compendium of six studies that were carried out to achieve the above-mentioned objectives. RESULTS: In studies 1, 2, and 5, IRBD patients differed from controls in several cognitive domains, namely attention and executive functions, mental processing speed, verbal memory, and semantic fluency. Besides, studies 1 and 5 identified for first-time impairment in facial recognition and visuospatial functions. Contrary to our hypothesis in study 1, we did not find significant correlations between cognitive performance and brain atrophy. However, there was coherence between the structural changes detected by MRI and neuropsychological impairment. For instance, IRBD subjects had facial recognition impairment and cortical atrophy in the fusiform gyrus, a region that is known to be critical for such function. In the same way, patients showed memory impairment and hippocampal atrophy, a core structure for learning and memory. On the other hand, study 2 evidenced the potential role of altered brain functional connectivity in IRBD’s cognitive impairment. We found a positive correlation between mental processing speed and temporoparietal functional connectivity in the IRBD group. In study 5, we reported visual form discrimination decline in IRBD relative to normal aging after less than two years. More importantly, we identified that visual form discrimination worsening over time was explained by progressive reductions of the cortical thickness in the superior parietal. CONCLUSIONS: The present Doctoral Thesis has identified new cognitive deficits in IRBD patients and has shed some light on its progression throughout the course of the disease. In particular, our work has shown the importance of VS/VP functions as a measure able to identify cognitive changes across time in IRBD, and its potential in identifying those patients with progressive neurodegeneration in posterior cortices. Besides, we have exhaustively described the characteristics of severe and early olfactory dysfunction in IRBD and described for the first time its neuroanatomical correlates. The neuroimaging data from this Thesis points towards an early implication of the temporal-occipital-parietal cortices in IRBD. Additionally, our longitudinal work has shown that IRBD patients have brain degeneration of the occipitoparietal and orbitofrontal cortex over time, in line with the degenerative changes reported in PD and DLB. Abnormalities in such regions are supported by the association between impaired cognition and both functional and structural brain changes. Cortical atrophy and disrupted cortico-cortical functional connectivity highlight that cortical degeneration and functional abnormalities already exist in patients with IRBD before they are diagnosed with DLB, PD, or MSA. Thus, suggesting that in prodromal PD, the alpha-synuclein pathology is already involving these structures earlier than would be expected according to the Braak degeneration model proposed form neuropathological data. Cortical atrophy and dysfunction may explain why some patients present neuropsychological impairment before the motor onset. We hope that our research will serve as a basis for further multicentric studies that confirm our findings, and to identify the optimal neuropsychological and neuroimaging markers for diagnostic, prognostic, and efficacy of future neuroprotective therapies.
MARC TEÒRIC: La present Tesi Doctoral estudia el trastorn del comportament del son REM (RBD, per les seves sigles en anglès) i la disfunció olfactiva com a biomarcadors de les alfa- Sinucleinopaties, com ara la malaltia de Parkinson (PD, per les seves sigles en anglès), la demència per cossos de Lewy (DLB, per les seves sigles en anglès) i l’atròfia multisistèmica (MSA, per les seves sigles en anglès). El RBD és una parasòmnia caracteritzada per la pèrdua d’atonia durant el son REM que provoca episodis on els pacients mostren moviments que reflecteixen el contingut dels seus somnis. El RBD que es presenta clínicament aïllat (IRBD, per les seves sigles en anglès), es considera un estadi prodròmic de les Sinucleinopaties, doncs s’ha vist que gairebé el 75% dels pacients després de 12 anys amb diagnòstic d’IRBD acaben sent diagnosticats de malaltia de Parkinson o parkinsonismes atípics. Concretament, en un estudi multicèntric el 56.5% dels pacients amb IRBD varen desenvolupar parkinsonisme com a primera manifestació, mentre que el 43.5% va presentar demència en primer lloc. La simptomatologia motora, la hipòsmia i el deteriorament cognitiu han demostrat ser els millors predictors de conversió en aquests pacients. En aquest context, la comunitat científica ha demostrat un interès creixent en definir els canvis cognitius associats a l’IRBD. Els estudis publicats fins ara conclouen que entre el 15-50% dels pacients tenen deteriorament cognitiu lleu. Malgrat l’evidència ben documentada sobre la presència d’afectació neuropsicològica en pacients amb IRBD, cal una recerca addicional que valori de forma aïllada i específica les funcions visuoespacials i visuoperceptives (VS/VP), ja que l’alteració d’aquestes funcions és característica del perfil neuropsicològic dels pacients amb PD i DLB. Pel que fa al declivi cognitiu al llarg de la malaltia, encara hi ha una gran incertesa. OBJECTIUS I HIPÒTESIS: Els objectius principals d'aquesta Tesi són: 1 Caracteritzar els substrats cerebrals estructurals i funcionals subjacents a l’RBD, així com relacionar aquestes troballes amb el rendiment cognitiu, 2 Estudiar la disfunció olfactiva com a biomarcador clínic i preclínic de les alfa- Sinucleinopaties, i la seva progressió al llarg de la malaltia, 3 Investigar la degeneració cerebral progressiva al llarg de l’IRBD i com aquests canvis es relacionen amb el declivi cognitiu. Les principals hipòtesis són: 1 L'IRBD es caracteritzarà per canvis específics en l'estructura cerebral i la connectivitat funcional que estarà associada a un deteriorament olfactiu i cognitiu, 2 Els pacients amb IRBD mostraran canvis cerebrals estructurals amb el pas del temps i un declivi cognitiu superior a l’observat en l’envelliment normal, 3 S'espera identificar reducció de la capacitat olfactòria en l'IRBD i la PD, així com que progressi lleugerament amb el curs de la malaltia. MATERIAL I MÈTODES: La present Tesi Doctoral consta de sis estudis realitzats per donar resposta als objectius esmenats anteriorment. CONCLUSIONS: La present Tesi Doctoral ha identificat nous dèficits cognitius en pacients amb IRBD i ha perfilar la seva progressió al llarg de l’evolució de la malaltia. En particular, el nostre treball ha demostrat la importància de les funcions VS/VP com a mesura capaç d’identificar els canvis cognitius a través del temps en l’IRBD i el seu potencial per identificar aquells pacients amb neurodegeneració progressiva en el còrtex posterior. A més, hem descrit de forma exhaustiva les característiques de la disfunció olfactiva precoç i greu de l’IRBD i hem descrit per primera vegada els seus correlats neuroanatòmics.

A doença de Alzheimer (DA) é caracterizada por declínio cognitivo e funcional progressivo e constitui-se como a forma mais prevalente de demência. O diagnóstico da DA é realizado atualmente, exclusivamente por meio de critérios clínicos. No entanto, a manifestação clínica da DA é precedida por um longo período assintomático, com depósito silencioso das proteínas tau e -amiloide no tecido cerebral. Evidências recentes demonstram que a mucosa olfatória, estrutura periférica e facilmente acessível também está acometida na DA, podendo representar um bom método de diagnóstico preciso e precoce desta moléstia. Os objetivos desse estudo consistem em correlacionar a prevalência das proteínas tau e -amiloide no epitélio olfatório (EO) com os estágios clínicos e neuropatológicos da doença e determinar a sensibilidade e especificidade dos achados no EO para o diagnóstico da DA em seus diferentes estágios de evolução. Para tal fim, é proposto estudo post-mortem, com avaliação de 92 tecidos cerebrais de indivíduos com idade igual ou superior a 50 anos, provenientes do Banco de Encéfalos Humanos do Grupo de Estudos em Envelhecimento Cerebral da Universidade de São Paulo, com coleta dos blocos de mucosa olfatória no momento da autópsia. A avaliação cognitiva foi realizada por meio de entrevista com informante de convívio próximo com o falecido, aplicando as escalas CDR e IQCODE. A avaliação neuropatológica do encéfalo e do EO foi realizada por meio de técnicas de . Os casos foram classificados mediante critério neuropatológico do CERAD e do estadiamento de Braak e Braak para DA. A presença de proteína tau e -amiloide no EO foi correlacionada com os parâmetros clínicos e neuropatológicos obtidos. A análise do EO da concha superior permitiu identificar as proteínas tau e -amiloide com sensibilidade alta, quando comparada com o diagnóstico neuropatológico (>80%) e clínico ( > 90%). Desta forma, a análise para as proteínas tau e - amiloide do epitélio olfatório pode representar um possível biomarcador para diagnóstico da DA
Alzheimer\'s disease (AD) is characterized by a progressive functional and cognitive decline and is considered the most prevalent type of dementia. AD is diagnosed exclusively on the basis of clinical criteria. However, clinical symptoms of AD are preceded by a long asymptomatic period, with silent deposition of tau and amyloid proteins in brain tissue. Recent studies demonstrate the same findings in the olfactory epithelium, a ready accessible structure which could contribute to the precise and early diagnosis of AD. The objectives of the current study were to correlate the prevalence of tau and amyloid proteins distributed in several areas of the olfactory epithelium with clinical and neuropathological criteria used for the diagnosis of AD and to determine the sensitivity and specificity of the olfactory epithelium involvement for the diagnosis of AD. Ninety-two individuals, belonging to the Brazilian Brain Bank of the Aging Brain Study Group from University of São Paulo, whose blocks of olfactory mucosa were collected during autopsy, were tested. Cognitive data were gathered through an interview with a knowledgeable informant, using the CDR (Clinical Dementia Rating Scale) and the IQCODE (Informant Questionnaire on Cognitive Decline in the Elderly) scales. Neuropathological examination was carried out on the basis of accepted criteria, using immunohistochemistry. Neuropathological classification of AD was performed in accordance with the CERAD criteria and Braak & Braak staging. The presence of tau and amyloid protein deposits in the olfactory epithelium was compared with clinical and neuropathological parameters. Immunostaining of olfactory epithelium from the superior turbinate was able to identify tau protein and amyloid-? with high sensitivity when compared with neuropathological scales ( > 80%) and clinical classification of CDR ( > 90%). Thus, immunohistochemistry for tau and amyloid proteins of the olfactory epithelium may represent a potential biomarker for early diagnosis of AD

L’odorat remplit des fonctions fondamentales dans le quotidien à travers son implication dans la détection de dangers, la prise alimentaire et les relations sociales. Par conséquent, la perte partielle (hyposmie) ou totale (anosmie) d’odorat a un impact significatif sur les conditions de vie, en affectant la santé physique et mentale des personnes touchées. Un nombre important de personnes sont concernées par les troubles de l’odorat, nombre qui est amené à croitre dans le contexte post-crise sanitaire du COVID-19 et en raison du phénomène de vieillissement des populations. Malgré leurs importantes prévalences et conséquences, les troubles de l’odorat sont mal pris en charge à cause d’un double manque d’accès au diagnostic et de traitements efficaces. Parmi les stratégies de remédiation olfactive, l’entrainement olfactif a émergé comme une piste prometteuse mais il reste à être optimisé car son efficacité apparait variable selon les études. L’entrainement olfactif pourrait également présenter des bénéfices plus larges sur le fonctionnement cérébral, qui sont actuellement peu explorés. Ce travail de thèse propose de nouvelles stratégies de diagnostic et de remédiation innovantes pour améliorer la prise en charge des troubles de l’odorat. Il explore également l’intérêt de l’entrainement olfactif en tant que stratégie de promotion globale du bien-vieillir au niveau cérébral. Nous avons développé une nouvelle génération de tests olfactifs connectés, permettant une évaluation rapide et facile des capacités olfactives, plus compatible avec une utilisation en routine clinique que les tests psychophysiques existants. Basé sur un score d’identification et d’évaluation de l’intensité de 8 odeurs, ce test permet d’identifier avec fiabilité les personnes souffrant d’anosmie de diverses origines. Afin d’améliorer l’efficacité de l’entrainement olfactif, nous avons développé un protocole assisté par une plateforme numérique, permettant d’améliorer le suivi de l’entrainement et l’adhérence du patient. Nous avons également testé si l’augmentation de la fréquence d'exposition aux odeurs ou le renouvellement des odeurs durant l’entrainement olfactif permettraient d’améliorer la récupération olfactive. Chez des patients post-COVID-19 présentant des déficits olfactifs persistants, un entrainement olfactif de 3 mois permet une amélioration de la perception olfactive ainsi que de la qualité de vie (notamment du plaisir alimentaire). Toutefois, l’augmentation du nombre d’odeurs senties quotidiennement ne semble pas améliorer l’efficacité de cet entrainement olfactif. Un entrainement basé sur l’utilisation de produits odorants du domicile se révèle également efficace mais nécessite une vigilance particulière vis à vis de l’adhérence du patient. Nous avons ensuite testé l’intérêt d’un entrainement olfactif avec renouvellement des odeurs, chez des participants âgés avec ou sans troubles de l’odorat. Les résultats suggèrent une meilleure récupération olfactive des participants dysosmiques ayant suivi cet entrainement pendant 4 mois, comparés à ceux ayant suivi à un entrainement avec une seule et même odeur. Enfin, nous avons évalué les potentiels bénéfices de l’entrainement olfactif avec renouvellement des odeurs sur la cognition des participants âgés, ainsi que sur le bien-être, la qualité de vie et l’appréciation alimentaire. Nos résultats ne montrent pas d’amélioration significative de la cognition, mais un bénéfice sur les symptômes dépressifs des participants âgés, avec ou sans troubles de l’odorat. L’analyse de l’activité du système noradrénergique ne montre pas de modification suite à l’entrainement olfactif, suggérant que celui-ci ne contribue pas aux mécanismes sous-tendant les bénéfices de l’entrainement. L’ensemble de ce travail ouvre donc de nouvelles perspectives pour le diagnostic et la remédiation des déficits olfactifs. Il suggère également que l’utilisation des odeurs pour promouvoir le bien-vieillir cérébral reste à approfondir
The sense of smell fulfills fundamental functions in everyday life, through its involvement in danger detection, food intake and social relations. Consequently, partial (hyposmia) or total (anosmia) loss of smell has a significant impact on living conditions, affecting the physical and mental health of those affected. A significant number of people are affected by smell disorders, a number that is set to grow in the post COVID-19 health crisis context and due to the phenomenon of ageing populations. Despite their significant prevalence and consequences, olfactory disorders are poorly managed, lacking access to diagnosis and effective treatment. Among olfactory remediation strategies, olfactory training has emerged as a promising approach but remains to be optimized as its efficacy appears to vary from study to study. Olfactory training could also have wider benefits on brain function, which are currently little explored. This thesis proposes new and innovative diagnostic and remediation strategies to improve the management of olfactory disorders. It also explores the interest of olfactory training as a strategy to promote cerebral well-ageing. We developed a new generation of connected olfactory tests, enabling rapid and easy assessment of olfactory capacities, more compatible with routine clinical use than existing psychophysical tests. Based on an identification and perceived intensity score of 8 odors, this test can reliably identify people suffering from anosmia of various origins. In order to improve the effectiveness of olfactory training, we developed a protocol supported by a digital platform, enabling improved training follow-up and patient adherence. We also tested whether increasing the frequency of odor exposure or renewing odors during olfactory training would improve olfactory recovery. In post-COVID-19 patients with persistent olfactory deficits, 3 months of olfactory training led to an improvement in olfactory perception and quality of life (including food enjoyment). However, increasing the number of odors smelled daily does not seem to improve the effectiveness of olfactory training. A training based on the use of scented products from home also proved effective but required particular vigilance with regard to patient adherence. We then tested the interest of olfactory training with odor renewal, in elderly participants with or without olfactory disorders. The results suggest better olfactory recovery in dysosmic participants who underwent this training for 4 months, compared with those who underwent training with a single odor. Finally, we assessed the potential benefits of olfactory training with odor renewal on the cognition of elderly participants, as well as on well-being, quality of life and food appreciation. Our results show no significant improvement in cognition, but a benefit on depressive symptoms in elderly participants, with or without smell disorders. Analysis of noradrenergic system activity showed no change following olfactory training, suggesting that it does not contribute to the mechanisms underlying the benefits of training. Taken together, this work opens up new perspectives for the diagnosis and remediation of olfactory deficits. It also suggests that the use of odors to promote better brain aging remains to be further explored

Schizophrenia is a strongly heritable multifactorial mental disorder that affects 1% of any population, but disease etiology is not yet fully understood. The limitations of using animal models, post-mortem brain studies and difficulty to obtain human brain biopsies have impeded the understanding of this disease from the biological perspective. There is a consensus, based on epidemiology and post-mortem human brains that schizophrenia is a neurodevelopmental disorder, but cellular and molecular mechanisms for this hypothesis are lacking. We have developed a model to address this using human olfactory neurosphere-derived (hONS) ‘stem’ cells, which were obtained non-invasively from human participants, expressed neural stem cell markers and allows comparative analyses of age- and sex-matched cell lines from patients diagnosed with schizophrenia with cell lines from healthy participants. Patient cells from various sources have a common dysregulation to gene pathway networks involved in axonal guidance, cell adhesion, cytoskeletal remodeling, focal adhesion signaling and reelin signaling - all important pathways that regulate cell migration. A high throughput live cell tracking technique was developed to unbiasly quantify cell motility in vitro. At baseline on uncoated tissue culture plastic (TCP), patient cells moved longer distances than control cells. Patient cells were unable to respond to their microenvironment when increasing concentrations of well-defined ECM proteins were coated on TCP cell culture surfaces. This was despite patient cells expressing the necessary cell surface integrin receptors measured by flow cytometry, which meant they have the capacity to bind and recognize all tested ECM proteins. Patient cells have consistently lower levels of cytoskeletal proteins in filamentous actin (F-actin) and stable microtubules, which became more evident when cultured on ECM proteins. Despite being able to adjust their cell sizes and shapes, patient cells were consistently smaller than control cells. Focal adhesion kinase (FAK) levels were consistently lower in patient cells and they were unable to manufacture enough focal adhesions of the correct sizes upon contact with ECM proteins. These findings highlighted a global deficit to cell migration in schizophrenia that was not limited to just Fibronectin but also on all ECM proteins, which were caused by disease-dependent intracellular deficits in patient cell cytoskeleton and focal adhesions. Reelin is an important brain ECM protein that orchestrates neuronal migration during neurodevelopment and its expression is reduced in post-mortem brain tissues from patients with schizophrenia. In this study, reelin protein levels were also reduced in patient hONS cells compared to control cells. Despite having the capabilities to bind to reelin through expression of key reelin signaling pathway accessory protein Dab1 and reelin-binding receptors ApoER2 and VLDLR, patient cells were unable to respond to extracellular reelin. Patient cells were unable to adjust their movement track lengths when tracked on surfaces coated with full length reelin. Similar to our previous findings on other ECM proteins, patient cells had lower levels of F-actin and stable microtubules, and were consistently smaller in size compared to control cells. Our findings suggested that non-responsive cell migration deficits were caused by defective focal adhesion responses to extracellular reelin. Instead of producing more focal adhesions that were larger in response to reelin, patient cells reduced both the densities and sizes of their focal adhesions at the peripheral region of the cell where lamellipodia forms. This was the first study to demonstrate a biological link between reelin signaling and schizophrenia, with a focus on the effects of reelin on cell migration. Published computer programs were used in silico to investigate the mechanistic components of cell migration and to further investigate reasons why patient cells were unable to respond to their ECM microenvironment. X-Y displacement coordinates for each individually tracked cell were analyzed by the DiPer and migration phase/cell turning analysis programs to compute cell movement variables in directionality, diffusivity, persistence and idling and turning. At baseline on TCP, patient cells did not move at the correct trajectories, failed to make directional changes, paused less and turned less when tracked for 24 h. When tracked on ECM proteins, it was difficult to pinpoint one explanation to explain our non-responsive cell migration findings as various mechanistic variables played a collective part to potentially give rise to the observed non-responsive cell migration phenotype. One explanation was that patient cells spent more time in persistent movement and were unable to make necessary changes to their directions. Another explanation was the unchanged cell pausing tendencies on Type I Collagen as a plausible explanation for observed non-responsive changes to motility on that ECM protein. The same in silico computational analyses showed that patient cells did not respond to extracellular reelin because they could not reduce the rate at which they changed the straightness of their movement trajectories. Patient cells were also unable to make well-timed changes to the direction of their movement trajectories by being more persistent in one direction for longer. Decreased densities and sizes of focal adhesions in patient cells upon contact with extracellular reelin resulted in patient cells not decreasing their turn angles, but instead made larger uncoordinated turns. The way that control cells responded negatively to extracellular reelin supported the hypothesis in the field that reelin acts as a “stop signal” for actively moving neuronal cells, which was disrupted in patient cells. In conclusion, this thesis has uncovered a novel non-responsive cell migration phenotype in schizophrenia, which was broadly relevant on different ECM proteins, to suggest consistent defects in how patient cells respond to their surrounding microenvironment. Findings from this thesis also showed that observed cell migration deficits were caused by differences to intracellular components such as cell cytoskeleton and focal adhesions, coupled to previously published findings that FAK, integrin and actin signaling pathways were all dysregulated in schizophrenia. Using an analogy of patient cells as a moving vehicle, our findings showed a global defect to many parts of this faulty “vehicle”, such as defective “ignition” in FAK signaling, uncoordinated “wheels” in the lamellipodia and consistent faults to the “clutch” mechanism in the focal adhesions. All working in concert to cause patient cells to move in a less coordinated fashion and unable to make controlled mechanistic changes to their movement, causing them to ultimately lose their way.
School of Environment and Sc
Science, Environment, Engineering and Technology
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INTRODUÇÃO: Apesar da fundamental importância da olfação para avaliação dos sabores dos alimentos ingeridos, percepção de vazamento de gases e de incêndios, sua avaliação clínica ainda não se encontra padronizada no Brasil. O Teste de Identificação do Olfato da Universidade da Pensilvânia (UPSIT) é um teste mundialmente utilizado e considerado por muitos como o padrão-ouro da avaliação olfatória. Originalmente em inglês, já foi traduzido para mais de 12 línguas. Esse trabalho se propôs a validar de forma inédita o UPSIT para outra cultura. O UPSIT versão em português foi validado para a população brasileira e tabelas normativas foram elaboradas para comparação do escore obtido segundo o sexo e idade do indivíduo. Secundariamente, procurou-se os fatores preditores de um melhor escore no teste. CASUÍSTICA E MÉTODOS: Estudo transversal realizado de dezembro de 2011 a agosto de 2012. A amostra utilizada foi não-probabilística por quotas e constituída por indivíduos presentes em uma instituição de atendimento público (Poupatempo São Paulo), de forma consecutiva, sem queixas olfatórias no dia do exame. Foi determinada a quota de 60 brasileiros(as) em cada faixa etária de cada sexo, a saber: 20-24, 25-30, 31-34, 35-40, 41-44, 45-50, 50-54, 55-59, 60-64, 65-69, 70-74, 75-79 e >= 80 anos. Após responderem questionário referente a dados demográficos e critérios de inclusão e exclusão do estudo, fizeram o UPSIT 782 indivíduos do sexo masculino e 796 do sexo feminino. Nos pacientes com idade igual ou superior a 65 anos foi realizado o Mini Exame do Estado Mental e se excluíram aqueles pacientes com escore inferior a 24 pontos pela possibilidade de quadro demencial. A versão do UPSIT aplicada nesse estudo foi resultado de dois estudos prévios para melhorar a aplicabilidade desse teste para a população brasileira. RESULTADOS: 1820 voluntários participaram do estudo, 1578 foram incluídos nas tabelas normativas. 242 foram excluídos no dia da entrevista por estarem com infecção das vias aéreas superiores, terem história de trauma crânio-encefálico, queixa de perda de olfato ou paladar e um escore menor de 24 no Mini Exame do Estado Mental. Verificou-se que entre os 1578 indivíduos analisados, o escore de UPSIT variou de 9 a 40, obtendo-se escore médio de 32,1 (desvio padrão: 5,3) e escore mediano igual a 33. Pela análise univariada (p < 0,01) e multivariada - regressão linear múltipla- (p < 0,05), observou-se que a idade, sexo, número de anos de estudo e renda mensal da família influíram no escore do teste. CONCLUSÕES: O UPSIT está agora validado para utilização na população brasileira. Disponibilizou-se tabelas normativas para avaliação olfatória e um modo rápido de obtê-las. Fatores de correção são necessários para uma perfeita equivalência entre as normas de todos os continentes, utilizando como padrão-ouro as normas do país onde a versão original do teste foi desenvolvida. Pior status econômico e educacional interferem negativamente na performance olfatória
INTRODUCTION: Despite the fundamental importance of olfaction to assess the flavors of food, perception of gas leakage and fire, its clinical evaluation is not yet standardized in Brazil. The University of Pennsylvania Smell Identification Test of the (UPSIT) is a test used worldwide and considered by many as the gold standard of olfactory assessment. Originally in English, it has been translated into more than 12 languages. This study aimed to validate the UPSIT for another culture in a novel form. The portuguese version of UPSIT Portuguese version was validated for the Brazilian population and normative tables were prepared to compare the score obtained by sex and age of the individual. Secondarily, we sought the predictors of a better score on the test. PATIENTS AND METHODS: Cross-sectional study conducted from December 2011 to August 2012. The sample used was a non-probabilistic by quotas and consisted of individuals present in a public service institution (Poupatempo São Paulo), consecutively, without olfactory complaints on exam day. We determined the quota of 60 Brazilians in each age group for each sex, as follows: 20-24, 25-30, 31-34, 35-40, 41-44, 45-50, 50-54, 55 -59, 60-64, 65-69, 70-74, 75-79 and >= 80 years. After answering a questionnaire about demographics and inclusion and exclusion criteria of the study, 782 males and 796 females did the UPSIT. In patients aged over 65 years was held the Mini Mental State Examination and excluded those patients with a score less than 24 points for the possibility of dementia. The version of the UPSIT applied in this study was the result of two previous studies to enhance the applicability of this test for the Brazilian population. RESULTS: 1820 volunteers participated in the study, 1578 were included in the normative tables. 242 were excluded on the day of the interview for being with upper airway infection, having an history of head trauma, complaining of smell or taste losses and a score below 24 on the Mini Mental State Examination. It was found that among the 1578 subjects analyzed, the UPSIT scores ranged from 9 to 40, yielding a mean score of 32.1 (SD: 5.3) and a median 33. By univariate analysis (p < 0.01) and multivariate analysis - multiple linear regression-(p < 0.05), it was observed that the age, sex, years of schooling and family monthly income influenced the test scores. CONCLUSIONS: UPSIT is now validated for use in the Brazilian population. Normative tables for olfactory assessment and a fast way to obtain them were demonstrated. Correction factors are needed for a perfect equivalence between norms of all continents, using as gold standard norms of the country where the original version of the test was developed. Worse economic and educational status interfered negatively in olfactory performance

Background: Patients with Panic Disorder (PD) direct their attention towards potential threat, followed by panic attacks, and increased sweat production. Onés own anxiety sweat odor influences the attentional focus, and discrimination of threat or non-threat. Since olfactory projection areas overlap with neuronal areas of a panic-specific fear network, the present study investigated the neuronal processing of odors in general and of stress-related sweat odors in particular in patients with PD. Methods: A sample of 13 patients with PD with/ without agoraphobia and 13 age- and gender-matched healthy controls underwent an fMRI investigation during olfactory stimulation with their stress-related sweat odors (TSST, ergometry) as well as artificial odors (peach, artificial sweat) as non-fearful non-body odors. Principal Findings: The two groups did not differ with respect to their olfactory identification ability. Independent of the kind of odor, the patients with PD showed activations in fronto-cortical areas in contrast to the healthy controls who showed activations in olfaction-related areas such as the amygdalae and the hippocampus. For artificial odors, the patients with PD showed a decreased neuronal activation of the thalamus, the posterior cingulate cortex and the anterior cingulate cortex. Under the presentation of sweat odor caused by ergometric exercise, the patients with PD showed an increased activation in the superior temporal gyrus, the supramarginal gyrus, and the cingulate cortex which was positively correlated with the severity of the psychopathology. For the sweat odor from the anxiety condition, the patients with PD showed an increased activation in the gyrus frontalis inferior, which was positively correlated with the severity of the psychopathology. Conclusions: The results suggest altered neuronal processing of olfactory stimuli in PD. Both artificial odors and stress-related body odors activate specific parts of a fear-network which is associated with an increased severity of the psychopathology.

Background: Patients with Panic Disorder (PD) direct their attention towards potential threat, followed by panic attacks, and increased sweat production. Onés own anxiety sweat odor influences the attentional focus, and discrimination of threat or non-threat. Since olfactory projection areas overlap with neuronal areas of a panic-specific fear network, the present study investigated the neuronal processing of odors in general and of stress-related sweat odors in particular in patients with PD. Methods: A sample of 13 patients with PD with/ without agoraphobia and 13 age- and gender-matched healthy controls underwent an fMRI investigation during olfactory stimulation with their stress-related sweat odors (TSST, ergometry) as well as artificial odors (peach, artificial sweat) as non-fearful non-body odors. Principal Findings: The two groups did not differ with respect to their olfactory identification ability. Independent of the kind of odor, the patients with PD showed activations in fronto-cortical areas in contrast to the healthy controls who showed activations in olfaction-related areas such as the amygdalae and the hippocampus. For artificial odors, the patients with PD showed a decreased neuronal activation of the thalamus, the posterior cingulate cortex and the anterior cingulate cortex. Under the presentation of sweat odor caused by ergometric exercise, the patients with PD showed an increased activation in the superior temporal gyrus, the supramarginal gyrus, and the cingulate cortex which was positively correlated with the severity of the psychopathology. For the sweat odor from the anxiety condition, the patients with PD showed an increased activation in the gyrus frontalis inferior, which was positively correlated with the severity of the psychopathology. Conclusions: The results suggest altered neuronal processing of olfactory stimuli in PD. Both artificial odors and stress-related body odors activate specific parts of a fear-network which is associated with an increased severity of the psychopathology.

Дисертація на здобуття наукового ступеня кандидата технічних наук за спеціальністю 05.11.17 – біологічні та медичні прилади і системи. – Національний технічний університет "Харківський політехнічний інститут", Харків, 2019. За відсутності сучасних доказових методів ольфактометрії доцільно розробляти методи та засоби респіраторно-ольфакторних порушень. У дисертаційній роботі подано розв'язання конкретного наукового завдання – розробка методів та засобів об'єктивного визначення респіраторно-ольфакторних порушень. На основі дослідження аеродинаміки носа на мікрорівні розроблено метод визначення ламінарного пограничного шару повітряного потоку у верхніх дихальних шляхах, що дозволяє за рахунок дослідження товщини пристінкової течії повітря відносно нерівномірності оболонки визначати патологічні ділянки носової порожнини за різних режимів дихання. Розроблено метод та засіб об'єктивної діагностики респіраторно-ольфакторних порушень, які дозволяють за рахунок визначення енергетичних характеристик носового дихання під час дії різних одорівекторів визначати на доказовому рівні відповідні пороги ольфакторної чутливості. Удосконалено метод визначення порогу ольфакторної чутливості, що дозволяє за рахунок аналізу форми циклограми носового дихання підвищити об'єктивність діагностики порушень нюхової чутливості або респіраторно-ольфакторних порушень.
The thesis of competition for the scientific degree of Candidate of Technical Sciences on specialty 05.11.17 – Biological and Medical Devices and Systems. – National Technical University "Kharkiv Polytechnic Institute", Kharkiv, 2019. In the absence of modern evidence-based methods of olfactometry, it is advisable to develop methods and means of respiratory and olfactory disorders. The dissertation is devoted to the solution of a specific scientific problem - the development of methods and means for the objective determination of respiratory and olfactory disorders. Based on the study of the aerodynamics of the nose at the micro level a method has been developed for determining the laminar boundary layer of the air flow in the upper respiratory tract, which makes it possible, by studying the thickness of the near-wall air flow relative to the unevenness of the mucous membrane, to determine the pathological regions of the nasal cavity in different breathing patterns. A method and means for objective diagnosis of respiratory and olfactory disorders have been developed, which allow, by determining the energy characteristics of nasal breathing under the action of various odorivectors, to determine the corresponding thresholds of olfactory sensitivity at an evidence-based level. The improved method for determining the threshold of olfactory sensitivity allows to increase the objectivity of diagnosing olfactory sensitivity disorders or respiratory olfactory disorders by analyzing the shape of the cycloramas of nasal breathing.

Дисертація на здобуття наукового ступеня кандидата технічних наук за спеціальністю 05.11.17 – біологічні та медичні прилади і системи. – Національний технічний університет "Харківський політехнічний інститут", Харків, 2019. За відсутності сучасних доказових методів ольфактометрії доцільно розробляти методи та засоби респіраторно-ольфакторних порушень. У дисертаційній роботі подано розв'язання конкретного наукового завдання – розробка методів та засобів об'єктивного визначення респіраторно-ольфакторних порушень. На основі дослідження аеродинаміки носа на мікрорівні розроблено метод визначення ламінарного пограничного шару повітряного потоку у верхніх дихальних шляхах, що дозволяє за рахунок дослідження товщини пристінкової течії повітря відносно нерівномірності оболонки визначати патологічні ділянки носової порожнини за різних режимів дихання. Розроблено метод та засіб об'єктивної діагностики респіраторно-ольфакторних порушень, які дозволяють за рахунок визначення енергетичних характеристик носового дихання під час дії різних одорівекторів визначати на доказовому рівні відповідні пороги ольфакторної чутливості. Удосконалено метод визначення порогу ольфакторної чутливості, що дозволяє за рахунок аналізу форми циклограми носового дихання підвищити об'єктивність діагностики порушень нюхової чутливості або респіраторно-ольфакторних порушень.
The thesis of competition for the scientific degree of Candidate of Technical Sciences on specialty 05.11.17 – Biological and Medical Devices and Systems. – National Technical University "Kharkiv Polytechnic Institute", Kharkiv, 2019. In the absence of modern evidence-based methods of olfactometry, it is advisable to develop methods and means of respiratory and olfactory disorders. The dissertation is devoted to the solution of a specific scientific problem - the development of methods and means for the objective determination of respiratory and olfactory disorders. Based on the study of the aerodynamics of the nose at the micro level a method has been developed for determining the laminar boundary layer of the air flow in the upper respiratory tract, which makes it possible, by studying the thickness of the near-wall air flow relative to the unevenness of the mucous membrane, to determine the pathological regions of the nasal cavity in different breathing patterns. A method and means for objective diagnosis of respiratory and olfactory disorders have been developed, which allow, by determining the energy characteristics of nasal breathing under the action of various odorivectors, to determine the corresponding thresholds of olfactory sensitivity at an evidence-based level. The improved method for determining the threshold of olfactory sensitivity allows to increase the objectivity of diagnosing olfactory sensitivity disorders or respiratory olfactory disorders by analyzing the shape of the cycloramas of nasal breathing.

Os Transtornos psiquiátricos acompanham a história da humanidade. Classificados em categorias distintas podemos observar que dentre todas as patologias que constituem os transtornos mentais e de comportamento, as doenças mais prevalentes são as doenças de Ansiedade e de Transtorno Depressivo Maior (TDM). Mesmo com muitos avanços nas neurociências, assim como na terapêutica (psicofarmacologia), ainda hoje a fisiopatologia e o desenvolvimento farmacológico são áreas que necessitam intenso estudo. Avanços recentes tem sugerido que drogas capazes de modular os sistemas glutamatérgico e purinérgico possuem potencial efeito neuromodulador, sendo promissores candidatos para o desenvolvimento de novas drogas com ação ansiolítica e/ou antidepressiva. Dessa maneira, o objetivo desta tese foi investigar o potencial efeito ansiolítico da guanosina (GUO) em modelos animais preditivos para o estudo da potencial atividade ansiolítica de novos compostos, assim como seu potencial efeito antidepressivo no modelo animal de depressão da Bulbectomia Olfatória Bilateral (OBX). Inicialmente, nossos resultados demonstram que a administração de GUO foi capaz de produzir um consistente efeito ansiolítico modulando os níveis de adenosina (ADO) e glutamato cerebral. Ainda, pela primeira vez, foi observado que a GUO per se promoveu uma diminuição da liberação de glutamato em preparações de sinaptosomas de hipocampo, um efeito dependente da ativação dos receptores A1 de ADO. Após a caracterização do potencial efeito ansiolítico da GUO, nosso objetivo foi avaliar o potencial efeito antidepressivo da GUO em um modelo animal com validade de face e de constructo, como o modelo da OBX. Contudo, após revisão da literatura em estudos que utilizaram o modelo da OBX, observou-se a necessidade de uma investigação a longo prazo, das principais alterações comportamentais e neuroquímicas induzidas pela OBX. Nossos resultados, mostram pela primeira vez, que camundongos submetidos a OBX apresentaram simultaneamente alterações comportamentais e neuroquímicas transitórias e de longa duração. Ademais, as evidências indicam que o hipocampo possui alta susceptibilidade aos danos induzidos pela OBX, visto que uma sinaptotoxicidade transitória, acompanhada de um duradouro desequilíbrio redox e aumento da resposta inflamatória foram observados. Por fim, o tratamento crônico com GUO foi capaz de reverter a maioria das alterações identificadas previamente como duradouras nos parâmetros comportamentais e neuroquímicos no modelo da OBX. Considerando os resultados neuroquímicos obtidos pelos diferentes protocolos de tratamento realizados neste estudo, novas hipóteses de mecanismos de ação exercidos pela GUO foram apresentadas, e mecanismos já estabelecidos foram reproduzidos. Por fim, de uma maneira geral os dados apresentados nesta tese reforçam a hipótese do envolvimento do sistema purinérgico nos transtornos psiquiátricos, e sugerem que a GUO apresenta uma potencial ação terapêutica para o tratamento destas doenças, abrindo assim novas perspectivas para elucidação dos mecanismos envolvidos na fisiopatologia da ansiedade e TDM.
Psychiatric disorder had accompanied the course of human history. Mental and behavioral disorders are classified in different categories and among all different psychiatric disorders; anxiety and major depressive disorder (MDD) are the most prevalents. Despite the substantial advances in our knowledge on the neurobiological bases of both anxiety and MDD, as well as in the therapeutic area (psychopharmacology), even today, the pathophysiology of these disorders as well as pharmacological development are still under investigation. Recent advances have suggested that drugs able to modulate glutamatergic and purinergic systems present a potential neuromodulatory effect, and are promising candidates for the development of new drugs with both anxiolytic and antidepressant effects. Thus, the aim of this work was to investigate the potential guanosine (GUO) anxiolytic-like effects in predictive animal models largely used to elucidate anxiolytic properties of new compounds, as well as investigate the potential GUO antidepressant effect in Olfactory Bulbectomy (OBX) model of depression. Initially, our results demonstrate that acute GUO administration was able to induce a consistent anxiolytic-like effect, by modulating the adenosine and glutamate cerebrospinal levels. Here, for the first time, it was observed that GUO per se was able to decrease the glutamate release in hippocampal synaptosome. After characterizing the potential anxiolytic-like effect promoted by GUO, our second goal was to evaluate the potential GUO antidepressant-like effect in an animal model with recognized face and construct validity as the OBX model of depression. However, given the lack of studies in the literature considering the time course of the behavioral and neurochemical changes after the depressive-like behavior onset induced by OBX we firstly characterize some important features regarding the OBX model. Collectively, mice submitted to the OBX model of depression and followed up to 8 weeks simultaneously presented transient and long-lasting deleterious effects in behavioral and neurochemical parameters. The evidences pointed that hippocampus was the most affected brain structure, since a transient hippocampal-related synaptotoxicity, accompanied by a long-lasting hippocampal imbalance in redox and inflammatory homeostasis were observed. Additionally, the neurochemical effects seem to strengthen our behavioral findings. Finally, chronic GUO treatment, similarly to the classical tricyclic antidepressant imipramine, was able to improve the long-term behavioral phenotype impairment induced by OBX, specifically improving behavioral performances that require cognitive functions, accompanied by reversion of hippocampal redox imbalance parameters, as well as in peripheral and central anti-inflammatory IL-10 release. Thus, in present study, the pre-clinical evaluation of GUO as a potential drug for treatment of the most prevalent psychiatric disorders (anxiety and MDD) presented promising results. Furthermore, additional GUO mechanisms of action were evidenced and new perspectives were established. Thus, the data presented in this thesis support the hypothesis of the involvement of the purinergic system in mood disorders, and suggest that GUO has a potential therapeutic activity for the treatment of psychiatric disorders.

ABSTRACT Background - Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer, primarily affecting about 6 million people worldwide. An early identification of PD is one of the main challenges in neurological research because to date, its diagnosis is still largely based on the clinical assessment of cardinal motor signs (bradykinesia, rigidity, resting tremor and postural instability) resulting by a progressive degeneration of dopaminergic neurons of the substantia nigra and locus coeruleus. However, impaired motor function appears when over 60% of the dopaminergic neurons are degenerated in the brain. In recent years, several evidence indicates that the onset of PD happens years to decades before the occurrence of classic motor symptoms. Pathological and imaging studies, for example, suggest that signs of nigrostriatal lesion can be detected 5–10 years before this clinical stage, and various observational prospective studies reveal that several non-motor symptoms (NMS) occur in this pre-diagnostic phase. Actually NMS such as olfactory impairment, cardiovascular dysautonomia such as orthostatic hypotension (OH) and rapid eye movement (REM) behaviour disorder (RBD) are currently being studied as features of prodromal PD and seem to be correlated to the early neuropathological process of disease. Beside these clinical manifestations, other biological alterations such as elevated oxidative stress and pro-inflammatory response have been involved in the cascade of events leading to degeneration of dopaminergic neurons. Recently, microRNAs (miRNAs) have been recognized as potent post-transcriptional regulators of PD-related gene expression. Consequently, the characterization of several NMS together with the assessment of molecular biomarkers linked to inflammation and oxidative damage, could be a potential methodological approach for the early identification of PD patients. Objectives - The main objective of my study was to explore potential novel diagnostic and prognostic biomarkers of PD. Specific study aims were, in patients with prodromal and established PD: a) to evaluate clinical markers such as olfactory and cardiovascular autonomic functions; b) to measure circulating mediators of oxidative stress and inflammatory response as early biomarkers of organ failure; c) to correlate biological findings with clinical functional alterations; d) to characterize specific circulating miRNA profiles in plasma samples. Methods - For this purpose, we recruited 15 patients with overt PD (Hoehn and Yahr stage I-III, on L-DOPA and dopamine agonists combination therapy), 11 subjects diagnosed with idiopathic RBD (iRBD) confirmed by lack of atonia during the REM sleep phase on polysomnography and 12 age- and gender-matched controls (CTRL). All enrolled subjects underwent the following assessments: total olfactory score (TOS) using Sniffin' Sticks Extended Test; autonomic function by measuring heart rate variability during deep breathing (DB) test, which expresses parasympathetic function, lying to standing (LS) test and the Valsalva manoeuvre (VM), that gives information about both sympathetic and parasympathetic function; antioxidant/oxidative stress mediators [glutathione (GSH), the most important endogenous scavenger, assessed in total and reduced form and in plasma and blood samples according to a high performance liquid chromatographic (HPLC) method; plasma malondialdehyde (MDA), a marker of lipid peroxidation, assayed by HPLC with fluorescence detection; 8-hydroxy-2-deoxyguanosine (8-OHdG), index of oxidative DNA damage, and 3-nitrotyrosine (3-NT), a stable end product of peroxynitrite oxidation, analyzed by commercial ELISA kits]; inflammatory response [plasma concentrations of tumor necrosis factor alpha (TNF) and interleukin 1-beta (IL1), the most important inflammatory cytokines, by ELISA commercial kits, while urine neopterin levels, a sensitive marker of cellular-mediated inflammation, were measured by an isocratic HPLC method]. Biochemical parameters were than correlated with clinical functional results. The miRNA profiling was performed in a subpopulation of the enrolled subjects (4 PD, 4 iRBD and 4 CTRL) by small RNA Sequencing, using Miseq sequencer (Illumina). The differentially expressed (DE) miRNAs analysis, based on the negative binomial distribution, was performed with DE Seq2 by performing three comparisons:1) iRBD versus CTRL; 2) PD versus iRBD; 3) PD versus CTRL. Subsequently, the relative expressions of specific miRNAs were validated in all study population by quantitative real-time (qRT) PCR using miScript PCR System kit (Qiagen). Results - A significant worsening trend was observed in total olfactory score, blood reduced GSH, LS and VM ratio and neopterin from the reference controls to iRBD and PD groups. In the multivariable ordinal logistic regression model, only low blood reduced GSH levels (p=0.037, OR=0.994; 95% CI 0.988 – 1.000), adjusted by history of hypertension, total olfactory score, LS ratio and VM ratio, were associated to PD status. Functional anosmia was similarly prevalent in iRBD (36%) and PD (33%) patients, but was absent in CTRL (p= 0.097). OH was more common among iRBD (73%) and PD (60%) than in controls (25%) (p=0.055), independently of antihypertensive treatment. A direct correlation was observed between total olfactory score and blood reduced GSH concentrations (R=0.034, p=0.037) and with VM ratio (R=0.43 p=0.015). Conversely, an inverse relation was found between total olfactory score and urine neopterin levels (R=-0.39 p=0.016). The results on circulating miRNA profiles found about 889 thousand sequenced reads mapped to mature miRNA sequences annotated in miRBase v21, by small RNA sequencing analysis. After data processing, no statistically significant DE miRNA was observed in the PD versus CTRL, whereas we found 33 DE miRNAs (18 downregulated, 15 upregulated, p-value <0.005) in the comparison between PD and iRBD and 6 (3 downregulated, 3 upregulated, p-value <0.005) in iRBD versus CTRL. Four common DE miRNAs (miR-101, miR-1260a, miR-142, miR15a) were dysregulated between the two different comparisons. In the PD patients, three miRNAs (miR-101, mir-142 and miR15a) were downregulated (Fold Change < -0.5) and only mir-1260a was upregulated (Fold Change > 0.5) with respect to iRBD. Conversely, miR-101, miR-142 and miR15a were upregulated and miR-1260a downregulated in iRBD compared to CTRL. The NGS results have not been validated by RT-PCR analysis till now because these miRNAs are poorly expressed in plasma. This condition makes very difficult, from a methodological point of view, their extraction. Discussion - The main findings of the present study are that reduced systemic antioxidant capacity is independently associated to overt PD and iRBD, a condition now established as prodromal PD, and correlates with olfactory and sympathetic dysfunction. Moreover, progressive cardiovascular autonomic dysfunction, expressed as altered sympathetic (VM ratio, OH) or parasympathetic (LS ratio) response to testing, is found from prodromal state to overt disease and correlates with olfactory dysfunction. Increased concentrations of neopterin, an inflammatory biomarker, are associated with worse olfactory dysfunction. The NGS analysis highlights a miRNA profiling in PD and iRBD subjects that needs to be verify, by changing and modifying the methodological approach for miRNA quantification. Conclusions - Reduced systemic antioxidant capacity is found in prodromal and overt PD and may represent, in association with olfactory loss and cardiovascular autonomic dysfunction, a useful additive biomarker of disease. Our pilot findings need to be confirmed in a larger population to establish their actual clinical value for an early diagnosis of PD.

La schizophrénie est une maladie psychiatrique très invalidante qui concerne près de 1% de la population. Bien que son étiologie soit toujours inconnue, elle est certainement multifactorielle et comprend une interaction entre prédisposition génétique et facteurs environnementaux. Il existe des traitements médicamenteux mais ils ne sont pas totalement efficaces, particulièrement pour la prise en charge des symptômes négatifs et des déficits cognitifs. Le développement de nouveaux traitements plus efficaces passe par l’amélioration des modèles animaux prenant en compte le caractère multifactoriel de l’étiologie de cette pathologie.Nous avons développé un modèle murin multifactoriel de schizophrénie innovant (modèle 3-hit) présentant une forte validité de construction. Pour cela, nous avons combiné une modification génétique (1er hit : délétion partielle du gène MAP6) avec un stress environnemental précoce (2nd hit : séparation maternelle de 24h au 9ème jour de vie) et une exposition tardive au THC durant l’adolescence (3ème hit : administration quotidienne de tétrahydrocannabinol à 8mg/kg du 32ème au 52ème jour).Dans un premier temps, nous avons montré une bonne validité d’apparence de ce modèle à travers des études comportementale, en imagerie et en électrophysiologie. En effet, au niveau comportemental les souris 3-hit présentent des symptômes de type négatif, des déficits cognitifs et une altération de la latéralité olfactive. Nous avons aussi montré un déficit d’inhibition du réflexe de sursaut, qui est un élément comportemental clef dans les modèles animaux de schizophrénie, car il est également utilisé en recherche clinique. Nous avons également observé certaines altérations morphologiques et fonctionnelles cérébrales caractéristiques de la schizophrénie comme une réduction du volume de l’hippocampe, une altération des fibres du corps calleux et un dysfonctionnement des systèmes de neurotransmission glutamatergique et GABAergique. Certains dimorphismes sexuels ont été également montrés dans nos études.Dans un deuxième temps, nous avons comparé les déficits des animaux 3-hit avec ceux d’autres modèles de schizophrénie développés au laboratoire. La caractérisation des effets de chaque facteur, indépendamment et en association, nous a permis de mettre en évidence un phénomène de synergie entre les facteurs et non une simple addition des déficits induits par chacun d’entre eux.Le modèle de schizophrénie 3-hit présente de bonnes validités de construction et d’apparence, il est maintenant nécessaire afin de parfaire sa caractérisation de tester sa validité pharmacologique
Affecting 1% of worldwide population, schizophrenia is a debilitating pathology. Whether the aetiology of schizophrenia remains unknown, its multifactorial aspect is conversely now well admitted, and certainly gathers genetic vulnerability and environmental factors. Actual treatments are still unmet, particularly for negative and cognitive symptoms. For a better translation from treatments design of schizophrenia to clinical efficiency, there is a crucial need to refine preclinical animal models that considers the multifactorial aspects of this disease.We developed a new murine multifactorial model of schizophrenia (3-hit), that possesses a strong construct validity. To this, we combined a genetic predisposition (1st hit: partial deletion of MAP-6) with an early postnatal stress (2nd hit: 24 h maternal separation at postnatal day 9), and a late cannabinoid exposure during adolescence (3rd hit: tetrahydrocannabinol THC from post-natal day 32 to 52; 8 mg/kg/day).First, we characterised a promising face validity through behavioural, imaging and electrophysiological studies. At behavioural level, we demonstrated that 3-hit mice displayed negative-like symptoms, cognitive deficits and altered olfactory laterality. Moreover, we showed a sensory motor gating deficit, that is a major translational clue for animal models of schizophrenia. Additionally, 3-hit mice displayed some characteristic morphological and functional impairments of the disease: reduced hippocampal volume, altered callosal fibres, glutamatergic and GABAergic neurotransmission dysfunctions. We moreover highlighted some sexual dimorphisms.Second, we compared deficits of 3-hit mice to those of others models of schizophrenia developed in our laboratory. Deficits induced by one factor, or combination of several factors, evidenced a synergistic effect, and not a simple addition between each of them.The 3-hit model therefore presents strong construct validity and promising face validity, encouraging to assess the pharmacological validity

Trabalho final do 6º ano médico com vista à atribuição do grau de mestre (área científica de neurologia) no âmbito do ciclo de estudos de Mestrado Integrado em Medicina.
BACKGROUND AND PURPOSE: Olfactory dysfunction is thought to be associated with Usher Syndrome (USH), although only few and controversial results are available. A recent animal study, along with other ciliopathies studies, provided support to the notion of olfaction underperformance in USH. We set out to report olfactory function for USH and both USH1 and USH2 genotypes. Olfactory bulb volumes, olfactory sulcus depths and olfaction-associated brain regions were also analysed. MATERIALS AND METHODS: Twenty-six controls with no previous olfactory deficit were age-and-sex-matched to 32 USH patients (11 USH1, 21 USH2). Morphometric Magnetic Resonance Imaging (MRI) and a butanol threshold test were used to evaluate brain structures and olfactory function, respectively. OB volumes and OS depths were manually measured by three operators (JR, AP, SF) using Osirix, with excellent intraclass coefficient tests. Averaged values across all measurements as well as brain regions’ volumes as segmented by Freesurfer were used for statistical analysis in SPSS. RESULTS: Olfactory thresholds were significantly higher in USH, Z = 3.508, p = 0.000452, and posthoc testing showed that this was mainly due to USH1 patients as compared to controls, p = 0.000184. OB volumes were not significantly different between groups, F(1,52) = 0,034, p = 0.855, and subgroups, F(2,50) = 0,798, p = 0.456. However, we did find butanol thresholds to be correlated with left OB volume for the USH1 subgroup alone (rs = -0,692, p = 0.018). OS depths across groups were found to be significantly different as shown by repeated-measures ANOVA, F(1,52) = 7,076, p = 0.01. Analysis of subgroups revealed a significant decrease for left OS depth, t(45) = 2,053, p = 0.047, only for USH2 patients (adjusted mean = 5.415 mm, DP = 0.548 mm) versus controls (adjusted mean = 7.586 mm; DP = 0.492 mm). As for brain regions, although differences were observed for a subgroup × gender analysis, F(2, 50) = 7.805, p = 0.001, the overall model for both group and subgroup analysis were not significant, F(1,52) = 1.980, p = 0.165 and F(2, 50) = 2.234, p = 0.118, respectively. CONCLUSIONS: The results provide evidence of olfactory dysfunction in patients with USH that correlates significantly with left OB volume specifically for the USH1 subgroup. Although olfaction is similar to controls, a decrease for left OS depth is present in the USH2 subgroup. CONTEXTO E OBJETIVO: Pensa-se que disfunção olfativa está associada ao Síndrome de Usher (USH), apesar de haver apenas um quantidade reduzida de artigos estar disponível, com bastante controvérsia.. Um estudo recente em animais, juntamente com outros estudos de outras ciliopatias, apoia a noção de défice olfativo em USH. Assim, colocamos como objetivo estudar a função olfativa para tanto USH e 2 dos seus genótipos (USH1 e USH2). Volumes dos bolbos olfativos (BO), profundidade dos sulcus olfativos (SO) e áreas cerebrais associadas com o olfato também foram analisadas. MATERIAIS E MÉTODOS: Vinte e seis controlos sem défice olfativo prévio foram emparelhados de acordo com a sua idade e género com 32 doentes USH (11 USH1, 21 USH2). Tanto a Ressonâcia Magnética (RM) morfométrica como um teste de limiar de butanol foram usados para avaliar estruturas cerebrais e função olfativa, respetivamente. Volumes dos BOs e profundidade dos SOs foram medidos manualmente por três operadores (JR, AP, SF) através do software Osirix, com excelentes testes de coeficiente intraclasse. Uma média dos 3 valores obtidos foram usados para cada medida assim como os volumes das áreas cerebrais medidos pelo Freesurfer foram usadas para análise estatística utilizando o SPSS. RESULTADOS: Os limiares olfativos foram significativamente superiores em USH, Z = 3.508, p = 0.000452, e testes posthoc demonstraram que isto se deve maioritariamente aos doentes USH1 quando comparados com controlos, p = 0.000184. Volumes do BO não foram significativamente diferentes entre grupos, F(1,52) = 0,034, p = 0.855, e subgrupos, F(2,50) = 0,798, p = 0.456. No entanto, os limiares de butanol revelaram-se negativamente correlacionados com o volume do bolbo olfativo esquerdo apenas para o subgrupo de USH1 (rs = -0,692, p = 0.018). A profundidade do SO revelou-se significativamente diferente entre grupos como demonstrado por uma ANOVA de medidas repetidas, F(1,52) = 7,076, p = 0.01. Análises dos subgrupos revelaram uma diminuição significativa para a profundidade de SO, t(45) = 2,053, p = 0.047, apenas para doentes USH2 (média ajustada = 5.415 mm, DP = 0.548 mm) versus controlos (média ajustada = 7.586 mm; DP = 0.492 mm). Quanto às regiões cerebrais, apesar das diferenças terem sido observadas numa análise subgrupo × género, F(2, 50) = 7.805, p = 0.001, o modelo geral para tanto a análise de grupo e de subgrupos não foi significativo, F(1,52) = 1.980, p = 0.165 and F(2, 50) = 2.234, p = 0.118, respetivamente. CONCLUSÃO: Os resultados demonstram evidência de disfunção olfativa em doentes com USH e que se correlaciona significativamente com o volume do BO esquerdo, em particular para o subgrupo USH1. Apesar do olfato ser semelhante aos controlos, uma diminuição da profundidade do SO esquerdo está presente no subgrupo USH2.

The aim of this project was to investigate whether reflex-like innate facial reactions to tastes and odors are altered in patients with eating disorders. Qualitatively different tastes and odors have been found to elicit specific facial expressions in newborns. This specificity in newborns is characterized by positive facial reactions in response to pleasant stimuli and by negative facial reactions in response to unpleasant stimuli. It is, however, unclear, whether these specific facial displays remain stable during ontogeny (1). Despite the fact that several studies had shown that taste-and odor-elicited facial reactions remain quite stable across a human’s life-span, the specificity of research questions, as well as different research methods, allow only limited comparisons between studies. Moreover, the gustofacial response patterns might be altered in pathological eating behavior (2). To date, however, the question of whether dysfunctional eating behavior might alter facial activity in response to tastes and odors has not been addressed. Furthermore, changes in facial activity might be linked to deficient inhibitory facial control (3). To investigate these three research questions, facial reactions in response to tastes and odors were assessed. Facial reactions were analyzed using the Facial Action Coding System (FACS, Ekman & Friesen, 1978; Ekman, Friesen, & Hager, 2002) and electromyography
Ziel dieses Projektes war es zu untersuchen, ob spezifische, mimische Reaktionen auf Geschmacks- und Geruchsreize bei Patientinnen mit Essstörungen verändert sind. Bei Neugeborenen rufen qualitativ verschiedene Geschmacksreize und Geruchsreize spezifische mimische Reaktionsmuster hervor. Diese Spezifität zeichnet sich infolge angenehmer Reize durch positive mimische Reaktionen und infolge unangenemher Reize durch negative mimische Reaktionen aus. Es ist jedoch unklar, ob diese spezifischen Reaktionsmuster während der ontogentischen Entwicklung stabil bleibe (1). Trotz der Befunde, dass geschmacks- und geruchsinduzierte mimische Reaktionen bei Erwachsenen relativ stabil bleiben, erlauben spezifische Forschungsfragen und verschiedene Methoden nur einen begrenzten Vergleich zwischen den Studien. Darüber hinaus könnten die gustofazialen Reaktionsmuster bei Patientinnen mit Essstörungen verändert sein (2). Diese Frage wurde jedoch bisher nicht untersucht. Weiterhin könnten Veränderungen in den mimischen Reaktionen bei essgestörten Patientinnen durch eine defizitäre Hemmungskontrolle bedingt sein (3). Zur Klärung dieser drei Fragestellungen wurden mimische Reaktionen auf Geschmacks- und Geruchsreize erfasst. Die Mimikanalyse erfolgte mit Hilfe des Facial Action Coding Systems (FACS, Ekman & Friesen, 1978; Ekman, Friesen, & Hager, 2002) und des Elektromyogramms

Research suggests that olfactory perception of patients with schizophrenia differs from healthy people. In the context of previously conducted research, the present thesis addresses the differences in the abilities of identification and discrimination of odours and assessment of perceived odour qualities (pleasantness, familiarity, intensity, edibility) between patients with schizophrenia or acute and transient psychotic disorders and healthy volunteers, taking into account gender differences; in addition, the differences in the relationship between identification of odours and evaluation of the perceived qualities of odours between patients and healthy persons were explored as well as relationship between the severity of negative symptoms and olfactory perception. In line with other studies, deficits were found in patients in identification and discrimination of odours and in the assessment of odour familiarity compared to healthy subjects, with no gender differences. There were no differences in the relationship between the identification of odours and assessment of the perceived qualities of odours between patients and volunteers, and association of olfactory abilities and the assessment of perceived qualities of odours with the severity of negative symptoms was found only for identification of...

Ziel: Das Ziel der vorliegenden Studie war einen Zusammenhang zwischen posttraumatischen Riechstörungen und Frontalhirnläsionen zu beweisen. Methode: Dafür wurden 51 Patienten mit posttraumatischen Riechstörungen und 50 Patienten mit Riechstörungen anderer Ätiologie untersucht. Der Grad der Riechstörungen wurde mit dem orthonasalen Testverfahren der Sniffin‘ Sticks sowie dem retronasalem Schmeckpulvertest eingestuft. Zusätzlich wurden neuropsychologische Tests (TMT-A, COWA, WCST, d2-R) durchgeführt, um Funktionen, die als typisch für die Frontallappen angesehen werden, zu überprüfen. Außerdem sollte jeder Patient den Fragebogen des Beck Depressions Inventars für eine Einschätzung seiner depressiven Tendenz beantworten. Ergebnisse: Es konnte in beiden untersuchten Patientengruppen kein Zusammenhang zwischen dem Riechvermögen und der neuropsychologischen Leistung festgestellt werden. Allerdings wurde in der posttraumatischen Patientengruppe eine signifikante, inverse Korrelation zwischen der Punktzahl des BDI und der Punktzahl im Diskriminationstest beobachtet. In der Kontrollgruppe bestand zwischen diesen Faktoren kein Zusammenhang. Schlussfolgerungen: Trotz der Ergebnisse dieser Studie kann ein Zusammenhang zwischen posttraumatischen Riechstörungen und Frontalhirnläsionen nicht ausgeschlossen werden, da u.a. die Funktionen der Frontallappen aufgrund ihrer Komplexität und Unspezifität schwierig zu messen sind. Außerdem waren die Traumata der Patienten sowie der klinische Verlauf sehr variabel und individuell. Frontalhirnläsionen können allerdings dramatische Einschnitte bedeuten, auf die klinisch besonders geachtet werden sollte. Olfaktorische Probleme können darüber hinaus zusätzlich die Lebensqualität stark beeinträchtigen und sollten daher ebenfalls Bestandteil der posttraumatischen Diagnostik sein.:Inhaltsverzeichnis 1 ABKÜRZUNGSVERZEICHNIS IV 2 EINLEITUNG 1 2.1 POSTTRAUMATISCHER RIECHVERLUST 1 2.2 FRONTALHIRN 3 3 ZIELSETZUNG DER STUDIE 9 3.1 HYPOTHESE 9 3.2 ERWARTETE ERGEBNISSE 10 4 MATERIAL UND METHODEN 10 4.1 PATIENTEN 10 4.2 METHODEN 10 4.2.1 RIECHTESTS 10 4.2.2 NEUROPSYCHOLOGISCHE TESTS 17 4.3 STATISTISCHE AUSWERTUNG 24 5 ERGEBNISSE 25 5.1 DESKRIPTIVE STATISTIK 25 5.1.1 PATIENTENKOLLEKTIV 25 5.1.2 POSTTRAUMATISCHE PATIENTEN 27 5.1.3 KONTROLLGRUPPE 28 5.2 KORRELATIONSPRÜFUNGEN 29 5.2.1 KORRELATION DER ERGEBNISSE DER RIECHTESTS MIT DEN ERGEBNISSEN DER NEUROPSYCHOLOGISCHEN TESTS 29 5.2.2 KORRELATION DER NEUROPSYCHOLOGISCHEN TESTS UNTEREINANDER 35 5.2.3 KORRELATION DER ERGEBNISSE DER RIECHTESTS MIT DER PUNKTZAHL DES BDI 36 6 DISKUSSION 39 6.1 METHODEN 40 6.1.1 RIECHTESTS 40 6.1.2 NEUROPSYCHOLOGISCHE TESTS 41 6.1.3 BECK DEPRESSIONS INVENTAR 43 6.2 ERGEBNISSE 43 6.2.1 ZUSAMMENHANG RIECHLEISTUNG MIT ERGEBNISSEN DER NEUROPSYCHOLOGISCHEN TESTS 43 6.2.2 ZUSAMMENHANG DISKRIMINATIONSLEISTUNG MIT PUNKTZAHL DES BECK DEPRESSIONS INVENTARS 48 7 AUSBLICK 50 8 ZUSAMMENFASSUNG 52 9 SUMMARY 53 10 ABBILDUNGSVERZEICHNIS V 11 TABELLENVERZEICHNIS VII 12 LITERATURVERZEICHNIS VIII 13 ERKLÄRUNG ZUR ERÖFFNUNG DES PROMOTIONSVERFAHRENS XXI 14 EINHALTUNG DER AKTUELLEN GESETZLICHEN VORGABE XXII 15 DANKSAGUNG XXIII 16 CURRICULUM VITAE XXIV 17 PUBLIKATIONEN XXVI

Olfactory impairment is considered an initial disturbance of several neurodegenerative diseases (NDs), including Parkinson’s disease (PD) and Alzheimer’s disease (AD). In addition, smell impairment precedes a decade, or even longer, the onset of motor or cognitive symptoms. Olfactory signals are detected by olfactory receptor proteins (ORPs) expressed in the cilia of olfactory receptor neurons (ONs). ONs are the distinctive cellular components of the peripheral olfactory epithelium (OE) and lie in the nasal vault. ONs axons pass the cribriform plate and reach the olfactory bulb (OB) where the olfactory stimuli are processed and sent to the superior nuclei of the CNS. Previous studies in AD and other neurodegenerative disorders have shown the presence of β-amyloid deposits in the OB, neurofibrillary tangles, as well as Lewy body pathology. OB represents the brain area earlier involved in the neuropathological process, decades before the development of clinical symptoms. Therefore, OB can be considered a target in the study of neurodegenerative diseases in their early molecular processes. Moreover, the OB of healthy subjects presents deposits of aggregated proteins confirming that these aggregates are deposited in a prodromal disease stage. Since the OB is an early accumulation site of aggregated proteins and the synapses derive from the ONs, it is possible that the first event of protein aggregation occurs in OE. ONs are directly exposed to the external environment including chemical/physical toxic injuries and such micro-environment predisposes to abnormal protein processing and folding (Sammeta and McClintock 2010). In addition, ONs and all other mature cell components have a half-life of three months and programmed apoptosis. The neural activity is maintained by a constant cellular turn-over, which is sustained by the basal stem cells. This regeneration process is persistent during the whole life of an individual, albeit with a decreasing rate with aging. Extensive scientific literature indicates the neuronal damage as the consequence of exposure to toxic injuries leading to neurodegeneration and ONs are a natural model of this noxious process (Lema Tomé, Tyson et al. 2013). The hypothesis of this pathological pathway is supported by several studies, in which aggregated forms of α-synuclein, tau and β-amyloid are detected in olfactory mucosa (OM) biopsies as well as in autoptic samples of patients with Parkinson’s disease (PD), Lewy body dementia (LBD), Frontotemporal dementia (FTD) and Alzheimer disease (AD) (Funabe, Takao et al. 2013) (Saito, Shioya et al. 2016) (Tabaton, Cammarata et al. 1991) (Talamo, Rudel et al. 1989) (Crino, Greenberg et al. 1995) (Arnold, Lee et al. 2010). In this study, we investigated for the first-time primary ONs sampled ex vivo using olfactory brushing (OBg) in normal subjects and patients with different neurodegenerative disorders. Because of its convenient location, OE is easily accessible and can be sampled to obtain the ONs in the tissue outer layer. This sampling method is harmless and non-invasive, bypassing potential artifacts due to post mortem specimens as well as avoiding the invasiveness of biopsy procedures. Recently, we showed that OBg procedure in Creutzfeldt-Jakob Disease (CJD) patients allows efficient OM sampling for the Real-Time Quaking-Induced Conversion (RT-QuIC) assay. We specifically amplified pathological prion protein (PrPSc) providing a diagnostic intra vitam test with sensitivity and specificity nearly to 100% (Orrú, Bongianni et al. 2014). For the purpose of our study, we firstly defined the phenotypic characterization of the human olfactory cells sampled with OBg from healthy subjects. Distinct antibodies were selected to analyze the olfactory epithelium cells: olfactory marker protein (OMP), neuron-specific class III β-tubulin (TUJ-1), protein gene product 9.5 (PGP 9.5), Pan-Cytokeratin (PCK). Secondly, we aimed to determine the expression patterns of the major misfolded proteins involved in the main neurodegenerative diseases. In particular, the selected proteins were: α-synuclein, APP/beta-amyloid, tau, and TDP-43. The identification of the expression patterns of these proteins in the ONs might provide information to understand the abnormal molecular mechanisms in the initial misfolding species involved in the pathological process. Moreover, in this study, we speculated on the subcellular locale where the protein aggregation may occur. Furthermore, by demonstrating the constitutive expression of the native NDs-associated proteins in the OE, we could assume that they may represent a potential template for triggering the aggregation process. Based on the immunocytochemistry analysis, we investigated the α-synuclein expression in patients affected by different synucleinopathies. In fact, α-synuclein misfolding and aggregation mechanisms are involved in the pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (LBD) and multiple system atrophy (MSA), which are all characterized by α-synuclein fibrils deposition (Spillantini, Schmidt et al. 1997). Finally, we analyzed the immunocytochemistry results in OM samples tested by α-synuclein RT-QuIC (α-syn RT-QuIC).

The rapid antidepressant effect of ketamine changed the direction of the research of potential antidepressants and its effect was also evaluated in this thesis. However, the main focus of this thesis is a new methodological approach to the research of depressive disorder. The main interest lies with the evaluation of automated monitoring of behaviour in this research. The first aim of this thesis was to evaluate the antidepressant effect of ketamine in the forced swimming test using software enabling automated monitoring of behaviour. The second aim was to meassure the change in phosphorylated Mammalian target of rapamycin (mTOR), using Enzyme-Linked ImmunoSorbent Assay (ELISA). The last, but the most important aim of this thesis was to implement the utilization of Phenotyper boxes in the automated behavioural evaluation of the olfactory bulbectomy model of depressive disorder and also evaluate the effect of ketamine in this model. Ketamine did not show an antidepressant effect in forced swimming test, however this observation could be influenced by chosen dose and mouse strain. Sensitivity of the test to chosen experimental protocol shows insufficient validity of this test. Observed change in level of phosphorylated mTOR corresponded with the behavioural results. Data collected from Phenotyper...