Introdução: A Doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum. Os sintomas motores são decorrentes da morte de neurônios dopaminérgicos da Substância Nigra mesencefálica e por inclusões intracitoplasmáticas de ?-sinucleína, os corpúsculos de Lewy (CL). A doença pode ser o resultado de fatores ambientais agindo sobre um indivíduo geneticamente susceptível. O objetivo desse estudo foi verificar a frequência de mutações no gene PARK8/LRRK2 em uma amostra de pacientes brasileiros portadores de DP e descrever as principais correlações clínicas encontradas nos pacientes com mutações. Metodologia: Estudo transversal baseado no protocolo padronizado pelo projeto LARGE-PD (Latin American Research Consortium on The Genetics of PD) aplicado em 282 pacientes com DP recrutados de ambulatórios especializados em Distúrbios do Movimento do Hospital das Clínicas de Ribeirão Preto/USP e do Hospital São Paulo/UNIFESP, entre os anos de 2007 e 2014. O material genético colhido foi enviado para Seattle, com análise genética realizada no laboratório do Dr. Cyrus Zabetian da Universidade de Washington. Resultados: Realizado pesquisa genética para o LRRK2 em 229 pacientes de 282 pacientes que preencheram o protocolo. Quatro (1,74%) pacientes foram positivos para a mutação. Nos casos de inicio precoce, a frequência foi de apenas um caso (2,43% - 1/41). Três pacientes tinham história familiar positiva para DP (3,7% - 3/81). A idade de inicio dos sintomas variou entre 38 e 55 anos. A mutação G2019S esteve presente em 1,31% (3/229). Foi encontrado também um caso de mutação para R1441C. Conclusões: O LRRK2 se mostrou um importante gene correlacionado a DP, tendo como principal mutação a G2019S. O início dos sintomas variou entre 38 e 55 anos, sempre unilateral, com boa resposta a Levodopa. / Introduction: Parkinson\'s disease (PD) is the second most common neurodegenerative disease. Motor symptoms are due to the death of dopaminergic neurons in the midbrain Substance Nigra and intracytoplasmic inclusions known as Lewy bodies (CL), rich in a protein called ?-synuclein. The disease can be the result of environmental factors acting on an individual genetically susceptible, multifactorial etiology. The aim of this study was to determine the frequency of mutations in the gene PARK8 / LRRK2 in a sample of brazilian patients with PD and describe the main clinical correlations in patients with mutations. Methodology: This is a crosssectional study based on a standardized protocol for LARGE-PD project (Latin American Research Consortium on The Genetics of PD) applied in 282 patients with PD recruited from specialized clinics in Movement Disorders seen at Hospital das Clínicas de Ribeirão Preto/USP and Hospital São Paulo/UNIFESP, between the years 2007 and 2014. The genetic material was sent to Seattle, and genetic analysis was performed in the laboratory of Dr. Cyrus Zabetian at the University of Washington. Results: Realized genetic research for LRRK2 in 229 patients of 282 patients who met the LARGE-PD protocol. Observed four (1,74%) patients positive for the mutation. In cases of early-onset, the frequency was only one case (2,43% - 1/41). Three patients had a family history of PD (3,7% - 3/81). The age of onset of symptoms in patients with mutations varied between 38 and 55 years. A total of PD patients with the DNA analyzed, G2019S was present in 1,31% (3/229). It was also found one case to mutation R1441C. Conclusions: The LRRK2 had great influence gene correlated with PD, the main mutation G2019S. The onset of symptoms varied between 38 and 55 years, always one-sided, with good response to levodopa.

Background: Neurodegenerative diseases (NDD) are very common in the aging population, of which Parkinson’s disease (PD) and Alzheimer disease (AD) are the two most common. Since the etiology of the neuronal death in these diseases remains unclear, currently no curative therapy is available. Traditional Chinese medicine (TCM) has been used to treat certain diseases, which based on their symptoms we now know that they are included PD and AD, for thousands of years. However, our pervious systematic review reports that the quality of current TCM clinical trials related to this area had limited internal validity due to methodological flaws and insufficient data reporting. Methods: This study includes two add-on double-blinded randomized controlled trials (RCT), PD full-scale study and AD pilot study. It aims to provide evidence for the efficacy and safety of two specific TCM decoctions, Jia-Wei-Liu-Jun-Zi Tang (JWLJZT) and Di-tan decoction (DTD) in treating PD and AD, respectively. These clinical trials follow the Consolidated Standards of Reporting Trials (CONSORT) as well as the International Conference on Harmonization guidelines on Good Clinical Practice (GCP). Also, this two RCT obtained the approval from the Human and Animal Research Ethics Committee of Hong Kong Baptist University before the study and registered on the Chinese Clinical Trial Registry. Result: In the PD trial, 111 idiopathic PD patients were randomly assigned to receive either JWLJZT or placebo for 32 weeks. Although there was not significant difference in the primary outcome of Movement Disorder Society Sponsored Revision of Unified PD Rating Scale (MDS-UPDRS) Part I total score (p = 0.216), significant improvements was observed in the secondary outcome of Non-motor symptom assessment scale for Parkinson’s disease (NMSS) total score (p = 0.019), subtype of mood/cognition (p = 0.005) and hallucinations (p = 0.024). In addition, post-hoc analysis showed a significant reduction in constipation (p < 0.001). On the other hand, 40 AD patients were randomly assigned to receive either DTD or placebo for 24 weeks in the AD trial. There was an improvement trend in the primary outcome of the cognitive subscale of Alzheimer’s Disease Assessment Scale (ADAS-cog) total score in the DTD group though the difference relative to the placebo group was not statistically significant (p = 0.315). No significant difference was found in the secondary outcomes. Adverse events were mild and comparable between treatment and placebo groups in both trials. Discussion: JWLJZT did show some improvement in non-motor symptoms, including mood, cognition, and constipation, in PD patients, while, DTD did show a reducing trend in the cognitive impairment based on rigorous RCT. Further study focusing on the effective dosage, pharmacologic mechanism of JWLJZT and DTD are needed to give a fuller picture as well as better support for using them in human being as a routine treatment. In fact, JWLJZT and DTD are the only two examples of TCM for treating NDD. These two clinical trials are served as examples of how to evaluate efficacy and safety of TCM for the treatment of various diseases using rigorous RCT methods and standard. Keywords: Randomized Controlled Trials, Parkinson’s disease, Alzheimer disease, Traditional Chinese medicine, Jia-Wei-Liu-Jun-Zi Tang, Di-tan decoction, Efficacy, Safety

A doença de Parkinson (DP) é uma patologia neurodegenerativa progressiva de causa desconhecida. A degeneração neuronal afeta vários grupos de neurônios no sistema nervoso central, mas de longe afeta muito mais os neurônios dopaminérgicos da pars compacta da substância nigra (SNc). Caracterizada por sintomas motores como bradicinesia, rigidez, tremor e instabilidade postural e sintomas não motores como depressão, ansiedade. O tratamento com o medicamento Levodopa está associado a efeitos colaterais, de ordem motora e não motora, em pacientes que o utilizam a longo prazo. Tais efeitos reduzem a qualidade de vida dos pacientes. Portanto esse estudo tem como principal objetivo analisar os sintomas de ansiedade em pacientes com DP idiopático que apresentavam flutuação motora (wearing - off, on e off), em tratamento com Levodopa, avaliar os sintomas da DP, principalmente o tremor e associar a ele a diminuição da ansiedade do paciente no momento em que se administra a medicação. Portanto, a coleta de dados ocorreu num primeiro contato com o paciente em off, ou seja, sem a Levodopa, em outros 3 momentos com o paciente em on. Foram aplicados a escala da Escala Analógica do Humor (VAMS), a Escala Unificada de Avaliação da Doença de Parkinson (UPDRS), Escala de Hoehn & Yahr (H&Y) e dois testes de Bradicinesia (Tapping, Pronação e supinação) para comparação evolutiva das atividades motoras e ansiedade do paciente em on e off. Concluímos que é comum a ocorrência de ansiedade em DP, que os níveis de ansiedade e a dificuldade motora são maiores quando o paciente está em off e que a administração do medicamento (Prolopa) reduz a ansiedade e melhora a função motora dos pacientes com DP. Esta melhora foi evidente nos testes de bradicinesia e na escala VAMS. / Parkinson\'s disease (PD) is a progressive neurodegenerative disease of unknown cause. The neuronal degeneration affects several groups of neurons in the central nervous system, but by far much more affects the dopaminergic neurons of the substance nigra pars compacta (SNc). Characterized by motor symptoms such as bradykinesia, rigidity, tremor and postural instability and non-motor symptoms such as depression, anxiety. Treatment with levodopa is associated with side effects, order motor and non - motor in patients who use the long term. These effects reduce the quality of life of patients. Therefore this study is meant to examine the anxiety symptoms in patients with idiopathic PD who had motor fluctuations (wearing - off, on and off), on treatment with levodopa, to assess the symptoms of PD, especially tremor and associate with it the decreased patient anxiety at the time administering the medication. Therefore, data collection occurred in a first contact with the patient off, without levodopa, 3 other times with the patient on. We applied the scale of the Analog Mood Scale (VAMS), the Unified Parkinson\'s Disease Rating Scale (UPDRS), Degree of Disability Scale of Hoehn & Yahr (H&Y) and two tests Bradykinesia (Tapping, pronation and supination) for comparison of evolutionary motor activity and anxiety of the patient on and off. Conclude that it is a common occurrence of anxiety in PD, levels of anxiety and motor difficulties are greater when the patient is off and that the drug (Prolopa) reduces anxiety and improves motor function of patients with PD. This improvement was evident in tests of bradykinesia and the VAMS scale.

Orientador: Elizabeth Maria Aparecida Barasnevicius Quagliato / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T09:41:15Z (GMT). No. of bitstreams: 1 Letro_GraceHelena_M.pdf: 1107376 bytes, checksum: 2610b83b9b3ecd9abf6796d76098a728 (MD5) Previous issue date: 2007 / Resumo: A dor é uma queixa muito freqüente na doença de Parkinson (DP) podendo preceder o diagnóstico da doença. O objetivo deste trabalho foi avaliar a dor num grupo de pacientes com DP. Utilizou-se a subescala II, III, IV (item - A) do UPDRS, a classificação segundo o estágio da doença Hoehn & Yahr, a escala de Schwab & England nos períodos On e Off , o questionário de dor Mc Gill, o inventário de depressão de Beck e o Mini- Exame do Estado Mental para exclusão de pacientes com escore menor que 20. Esta casuística foi constituída por 20 mulheres e 30 homens. A média de início dos sintomas foi de 54,84 anos +/- 9,54. A média de tempo de doença foi de 8,06 anos +/- 4,77. A dor foi relatada durante a anamnese por 27 pacientes; 22 (44%) no período de exacerbação dos sintomas (Off) e 5 (10%) pacientes no período de melhora e exacerbação dos sintomas (On e Off). Os dados demonstraram que a dor na DP apresentou uma freqüência de 54% nos 50 pacientes avaliados. O aumento da dor no período Off foi relatado pelos pacientes, sendo a rigidez um fator importante para o aparecimento da mesma. Não houve associação entre dor:- e tremor no período Off, o tempo de doença, o estágio Hoehn & Yahr, a escala de Schwab & England , a depressão, a discinesia. Observou-se melhora da dor com levodopa em 16 (59,26%) dos pacientes / Abstract: Pain is a very frequent complaint in the Parkinson's disease (PD) being able to precede the diagnosis of the disease. The objective of our work was to evaluate pain in a group of patients with PD. To evaluate pain were used the subscale II,III,IV ( item-A) of the UPDRS, the Hoehn & Yahr staging, Schwab and England ADL Scale in the period On and Off, the Questionnaire of pain McGill , the Inventory of depression of Beck and the Mini Examination of the Mental State for exclusion of patients with lesser score that 20. We evaluate 50 patients with PD, 20 women and 30 men. The average of beginning of the symptoms was of 54, 84 years +/_ 9, 54.The mean of duration of disease was of 8, 06 years+/_4, 77. Excluding the secondary illnesses, pain was told by 27 patients during of the clinical history; 22 (44%) in the aggravation period and 5 (10%) in the period of improvement and aggravation of the symptoms (On and Off). We data had demonstrated that pain in the PD presented a frequency of 54% in the 50 evaluated patients. The increase of pain in the off period was told by our patients, being the rigidity an important factor for the appearance of the same one. There wasn¿t association between pain: and tremor in the Off period, duration of disease, Hoehn & Yahr staging, scale of Schwab & England, the depression, the diskynesia. Our data had confirmed the improvement of pain with the use of levodopa in 16 (59, 26%) patients / Mestrado / Neurologia / Mestre em Ciências Médicas

Orientador: Elizabeth Maria Aparecida Barasnevicius Quagliato / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T15:34:29Z (GMT). No. of bitstreams: 1 Macedo_LydiannaSilveirade_M.pdf: 1373438 bytes, checksum: f800e333bfb6e73b680594669c7d8399 (MD5) Previous issue date: 2009 / Resumo: Os problemas da mobilidade, como quedas e congelamento da marcha, têm um impacto devastador na vida de pacientes com Doença de Parkinson (DP) e não apresentam resposta a levodopa. Sabe-se que as pistas visuais e auditivas melhoram o inicio e a geração da seqüência da marcha e funcionam como via alternativa à programação interna dos núcleos da base que se encontra alterada. Sugere-se, com isso, que esses sinais facilitem o processo de preparação motora em pacientes parkinsonianos, reduzindo o número e a gravidade dos episódios de congelamento. O objetivo desse estudo foi avaliar a influência da utilização de pistas visuais e auditivas, durante a terapia em grupo, na reabilitação de pacientes parkinsonianos com sintomas de congelamento. Participaram do estudo 20 pacientes com DP, que foram divididos em 4 grupos, os quais receberam dezesseis sessões de terapia utilizando pistas visuais e auditivas. Foram avaliados o número e a gravidade dos episódios de congelamento, a caracterização clínica da doença, função motora, realização das atividades de vida diária, equilíbrio estático e dinâmico, mobilidade funcional, velocidade da marcha, função cognitiva e qualidade de vida. Os pacientes foram avaliados em 4 momentos: contato inicial, antes do início da terapia em grupo (dois meses após o contato inicial), após a última sessão de terapia em grupo e 2 meses após o final da intervenção (follow-up). A pesquisa resultou em melhora significativa do número e da gravidade dos episódios de congelamento, função motora, atividades de vida diária, caracterização clínica da doença e qualidade de vida. A conclusão desse estudo foi que a terapia em grupo com utilização de pistas visuais e auditivas influenciou no sintoma de congelamento dos pacientes com doença de Parkinson, levando a uma redução do número e da gravidade dos episódios, havendo manutenção dessa melhora por um período de 2 meses após a intervenção proposta. / Abstract: Mobility's problems, such as falls and freezing of gait, have a devastating impact on patients with Parkinson's disease's (PD) lives and have no response to levodopa. It is known that the auditory and visual cues improve the generation and beginning of motion sequence and function as alternative to the internal programming of the changed base's nuclei. It is suggested, thus, that they facilitate the motor preparation process in Parkinsonian patients, reducing the number and severity of freezing's episodes. This study's purpose was the evaluation, while in group therapy, of influence of visual and auditory cues use in the rehabilitation of Parkinsonian patients with freezing symptoms. Twenty patients with PD were evaluated. They were divided in four groups, which received sixteen visual and auditory cues therapy sessions. The number and severity of freezing episodes, the clinical disease characterization, motor function, performance on daily living activities, static and dynamic balance, functional mobility, movement speed, cognitive function and quality of living had been evaluated. Patients were evaluated in 4 stages: initial contact, before the therapy group (two months after the initial contact) after the last session of group therapy and 2 months after the intervention (follow-up). The search resulted in significant improvement in the number and severity of freezing episodes, motor function, daily living activities, clinical characterization of disease and quality of living. The conclusion was that the treatment group with use of visual and auditory cues influenced symptom of freezing in patients with Parkinson's disease, leading to a reduction in the number and severity of episodes, with maintenance of improvement for a period of 2 months after the proposed intervention. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

O objetivo do presente estudo foi elaborar um teste de divisão de atenção envolvendo tarefas funcionais da marcha, controle postural estático e dinâmico e controle manual e aplicar este teste em adultos jovens, idosos e indivíduos com DP em estágio iniciais e moderados de evolução da doença. Trata-se de um estudo transversal realizado na Associação Brasil Parkinson, São Paulo. Participaram do estudo 20 adultos jovens, 17 idosos e 47 indivíduos com diagnóstico de DP, 16 no estágio I na escala de estadiamento de Hoehn Yahr, 15 no estágio II e 16 no estágio III. O desempenho dos 84 participantes foi verificado durante a execução de quatro tarefas denominadas de funcionais: marcha, controle postural dinâmico, controle postural estático e uma de controle manual. Também foi analisado o desempenho de oito tarefas específicas, quatro de características motoras e quatro cognitivas. As doze tarefas, funcionais e específicas, foram avaliadas quando executadas isoladamente (condição chamada de tarefa simples (TS), e também em dupla tarefa, quando uma tarefa funcional foi executada simultaneamente a uma tarefa específica motora (dupla tarefa motora (DTM) e também associada a uma tarefa específica cognitiva (dupla tarefa cognitiva (DTC). Nas condições de dupla tarefa o desempenho de ambas as tarefas foi mensurado. A análise estatística foi realizada por meio de ANOVA de medidas repetidas e pós hoc teste de Tukey para a verificação de possíveis diferenças de desempenho entre jovens e idosos, idosos e pacientes com DP, e entre pacientes nos diferentes estágios da DP. Na comparação entre jovens e idosos, as quatro tarefas funcionais propostas mostraram-se sensíveis para identificar as diferenças associadas ao processo de envelhecimento, em especial a marcha e a tarefa de controle manual. Já entre idosos e indivíduos com DP, a tarefa mais sensível para identificar diferenças foi a de controle manual, onde os indivíduos com DP apresentaram desempenho inferior em todas as condições. Na comparação entre os indivíduos com DP no estágio I, II e III a menos sensível foi a de controle manual, que não identificou prejuízos entre os estágios iniciais da doença (I e II). Com intuito de verificar o impacto global da associação de tarefas, foi analisada a soma dos prejuízos da divisão da atenção para a tarefa funcional e a tarefa específica motora e cognitiva, chamado de custo total motor e custo total cognitivo. Observamos diferentes padrões de resultados: a combinação entre a tarefa de controle postural estático e uma tarefa motora foi a condição mais sensível para identificar as alterações do processo de envelhecimento na habilidade de dividir a atenção, entre idosos e indivíduos com DP nenhuma das condições foi capaz de identificar diferenças e entre os estágios da DP, o custo da divisão da atenção entre a tarefa de controle postural estático e uma tarefa cognitiva foi a condição mais sensível. Conclui-se que o teste de avaliação em divisão de atenção proposta mostrou ser um instrumento de boa aplicabilidade, baixo custo e sensível para a identificação de alterações decorrentes do processo de envelhecimento, prejuízos associados à DP e sua progressão / The aim of this study was to develop a testing division of tasks involving attention functional gait, postural control static and dynamic control manual and apply this test in young adults, seniors and individuals with PD in early and moderate stage of the disease. This is a cross-sectional study in Associação Brasil Parkinson, São Paulo. Study participants were 20 young adults, 17 seniors and 47 individuals diagnosed with PD, 16 in stage I in the scale of Hoehn Yahr staging, 15 in stage II, 16 stage III. The performance of 84 participants was observed during the execution of four functional tasks named: gait, postural control, dynamic, static postural control and manual control. We also analyzed the performance of eight specific tasks, four motor and four cognitive characteristics. The twelve tasks, and specific functional, were evaluated when performed alone (called simple task (ST), and also in dual task, when a functional task was performed simultaneously with a specific motor task (dual motor task (DMT) and also associated with a specific cognitive task (dual cognitive task (DCT). Under the conditions of dual task performance of both tasks was measured. Statistical analysis was performed using repeated measures ANOVA and post hoc Tukey test for checking possible performance differences between young and old, elderly and PD patients, and between patients in different stages of PD. Comparing young and old, the four functional tasks proposals were sensitive to identify differences associated with aging, particularly walking and manual control task. Among elderly and individuals with PD, the most sensitive task was to identify differences in manual control, where individuals with PD showed poorer performance in all conditions. Comparing individuals with DP in stage I, II and III was the least sensitive to manual control, which did not identify losses from the early stages of the disease (I and II). Aiming to verify the impact of the global association task, we analyzed the sum of losses of the division of attention to the task and the task specific functional motor and cognitive, called the total cost of motor and cognitive total cost. observed different patterns of results: the combination of static postural control task and a motor task was the most sensitive condition to identify the changes of aging on the ability to divide attention among elderly individuals with PD and none of the conditions was able to identify and differences between stages of PD, the cost of the division of attention between the task of static postural control and a cognitive task was the most sensitive condition. As conclusion that the assessment test in proposed division of attention shown to be an instrument of good applicability, inexpensive and sensitive for identifying changes resulting from the aging process, and losses associated with DP its progression

The deposition of amyloid in the central nervous system is associated with prevalent neurological disorders such as Alzheimer's and Parkinson's disease. This thesis studies the mechanisms and prevention of amyloid formation in vitro. We specifically focus on Parkinson's disease associated α-synuclein (α-syn). Using novel labeling methods we introduce NMR observable labels onto lysosomal protein glucocerebrosidase (GCase), a leading cause of Parkinson's disease. By introducing NMR active labels we are able to study GCase dynamics and screen potential drug therapeutics (chapter 3). Furthermore, we analyze the three way interaction between GCase, α-syn and lipids. We conclude that GCase is able to effectively chaperone α-syn under lysosomal conditions, both preventing amyloidogenesis and destabilizing mature amyloid fibrils (chapter 4). Additionally, a model chaperone-aggregate system is investigated to gain insight into the mechanisms of small heat shock protein chaperoning, and how such mechanisms prevent aggregation (chapter 5). Finally, a high resolution crystal structure of RNA editing enzyme Cid1 is presented, whilst not directly linked to aggregation, many of the techniques used in this thesis were first developed on Cid1 (chapter 7). Together, we utilize NMR, X-ray crystallography, electron microscopy and native mass spectrometry to elucidate aspects of protein aggregation mechanisms and prevention.

Increasing evidence has shown that abnormal metabolic phenotypes in body fluids reflect the pathogenesis and pathophysiology of Parkinson's disease (PD). However, the relationship between metabolic phenotypes and PD is not fully understood. Mass spectrometry (MS) based metabolomics is a powerful technique, which was frequently used for the sensitive and reproducible detection of hundreds to thousands of metabolites in biofluid samples.. Here we developed and performed MS-based metabolomics studies involving hundreds of human urine samples with data acquired from multiple analytical batches for surveying potential biomarkers of PD. A new software statTarget was developed and introduced. Protocols for liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) were developed, including sample preparation, data acquisition, quality controls, quality assurance and data analysis. Urinary metabolites from a total of 401 clinical urine samples collected from 106 idiopathic PD patients and 104 normal control subjects were profiled by using LC-MS. Quality control (QC) strategy has been performed in MS-based metabolomics for high reproducibility and accuracy of MS data. GC-MS with methyl chloroformate (MCF) derivatization was used for profiling highly polar metabolites in patients with early-, middle- and advanced-stage PD. Our study revealed the significant correlation between clinical phenotypes and urinary metabolite profiles. Comprehensive metabolomics was successfully developed with the goal of identifying urinary metabolite markers that can be used for evaluating the development of PD. A group of 18 metabolites have shown not only a high discriminating ability for the early-stage PD patients but also accurately distinguished the middle- and advanced- stages patients from control subjects. For the evaluation of PD, 18 metabolites showed good potential as metabolite markers with related metabolic pathway variations observed in branched chain amino acid metabolism, glycine derivation, steroid hormone biosynthesis, tryptophan metabolism, and phenylalanine metabolism.. We have further performed targeted analysis of potential biomarkers by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and GC-MS. The UPLC-MS/MS method was developed and optimized for detecting the concentration variation of metabolites in tryptophan metabolism for alpha-synuclein over-expressed flies (Parkinson's disease model). The altered tryptophan metabolism was proved as one of the common metabolite signatures between PD patients and alpha-synuclein over-expressed fly model of PD, and thus may be used for developing potential markers of the disease and evaluating the efficacy of novel therapeutic agents. An asymmetric labeling strategy and positive chemical ionization gas chromatography-tandem mass spectrometry (PCI-GC-MS-MS) approach was developed for the determination of non-amino organic acids and amino acids, as well as short chain fatty acids. Carboxylic and amino groups could be selectively labelled by propyl and ethyl groups, respectively. The specific neutral losses of C3H8O (60 Da), C3H5O2 (74 Da) and C4H8O2 (88 Da) were useful in the selective identification for qualitative analysis of organic acids and amino acid derivatives. The developed PCI-GC-MS/MS method showed good reproducibility and linear range.. In summary, metabolomics study has its inherent advantage in the characterization of biomarkers for the development of PD and may bring new scientific knowledge as well as impact on the progression of PD and other related neurodegenerative diseases.

Dopamine (DA) is a key striatal neuromodulator central to normal functioning of the basal ganglia. Identifying and characterizing circuits governing striatal DA transmission is necessary for understanding DA involvement in adaptive behaviour and pathology. Properties of evoked striatal DA release can be examined using fast-scan cyclic voltammetry at carbon fibre microelectrodes, a technique enabling live monitoring of transmitter release events with sub-millisecond resolution. Experimental work presented in this thesis employed this approach to study regulation of striatal DA by acetylcholine (ACh) in health and disease in acute brain slices. Synchronous activity in a small population of striatal cholinergic interneurons (ChIs) was previously shown to directly drive striatal DA release. Here using optogenetic approach I explore physiological relevance of ChI-evoked drive of striatal DA by examining whether corticostriatal and thalamostriatal afferents to ChIs can trigger ACh-evoked DA events. Following floxed vector injections in motor cortex or caudal intralaminar thalamus of CaMK2a-Cre mice I examine the properties of evoked DA upon light activation of channelrhodopsin-2-transduced inputs to striatal ChIs. These experiments revealed that both cortical and thalamic afferents are capable of driving ACh-evoked DA release, but operate using a different complement of post-synaptic ionotropic glutamate receptors and display distinct release recovery profiles. I further explore if rebound excitation in a population of striatal ChIs could drive DA events by examining whether ACh-evoked DA release follows optical inhibition of striatal ChIs selectively expressing hyperpolarizing halorhodopsin 3.0 or archaerhodopsin 3.0 in ChAT-Cre mice. This work showed that hyperpolarizing ion pumps were not successful in triggering ChI-evoked DA release. I also investigate whether cholinergic brainstem innervation of striatum could contribute to or drive ACh-evoked striatal DA events in ChAT-Cre rat, concurrently showing that ChI-evoked DA release is not a species artefact, and is present in mouse and rat alike. Current results also suggest that cholinergic brainstem afferents do not drive or contribute to striatal ACh-evoked DA events. Close interaction between DA and ACh systems further indicates that ACh could impact dopaminergic dysfunction. To explore this I examined the state of ACh transmission in a mouse model of Parkinson’s disease overexpressing human wild type alpha–synuclein protein. These animals present with impaired striatal DA release from young age, but DA deficits could be mediated by changes in ACh tone. Here I show that impaired striatal DA release is the results of primary DA axon dysfunction, although in ventral striatum DA release deficits could be partially compensated by increased ACh tone at nicotinic receptors. I further show that the functional state of muscarinic ACh receptors in not altered following decreased DA transmission, although the data from aged animals suggest that alpha–synuclein-dependent changes in vesicle handling could contribute to impaired DA releasability. Finally, I show that vesicle handling may indeed be altered in this mouse model as impaired DA release is evident with short stimulation protocols, while with prolonged depolarization of DA axon terminals alpha–synuclein-overexpressor mice are better able to sustain evoked DA release. Overall, the main body of work presented in this thesis examined the processes regulating striatal DA transmission via ACh system. In particular, I show that ChI-evoked drive of striatal DA release can be recruited physiologically and further establish that changes in ACh transmission are not the primary drivers of impaired DA releasability in a mouse model of Parkinson’s disease overexpressing human alpha–synuclein protein.

Doctor of Philosophy / Department of Anatomy and Physiology / Mark L. Weiss / Umbilical cord matrix or Wharton’s Jelly is a mucous connective tissue ensheathing the cord blood vessels and contains mesenchymal-like stem cells. Previously, we have shown that pig umbilical cord matrix stem (pUCMS) cells transplanted into normal rat brain were recovered up to 6 weeks post-transplantation, where a sub-population of pUCMS cells exhibited neuronal morphology and expressed a variety of neuronal markers. Here, approximately 150 pUCMS cells were transplanted into non-immunesuppressed rats that previously received a brain lesion by neurotoxin, 6-hydroxydopamine (6-OHDA), which specifically affects midbrain dopaminergic neurons, leading to pathologic findings similar to that of Parkinson’s disease (PD). The pUCMS cells proliferated up to 8 weeks post-transplantation and there was a significant increase in the percentage and number of pUCMS cells expressing tyrosine hydroxylase (TH), which is a marker for dopaminergic cells. We conclude that 1. Xenotransplants of pig UCMS cells are not rejected by rats at least up to 8 weeks after transplantation and 2. The pig UCMS cells proliferate and differentiate after transplantation into PD model rats. The surface antigen and gene expression profile of human umbilical cord matrix stem (hUCMS) cells resemble that of mesenchymal stem cells. Apomorphine-induced rotatory behavior was used to analyze the motor deficits of the PD model rats. In different experiments 1000, 2500 and 25000 hUCMS cells were transplanted into the brain of non-immunesuppressed PD model rats. There was a dose-dependent decrease in apomorphine-induced rotations; the maximum benefit was found in the rats that received 1000 hUCMS cells. The graft cells were recovered at 2 days and 1 week, but not at 6, 10 or 12 weeks post-transplantation. Quantitative assessment of host TH-positive midbrain dopaminergic neurons revealed a positive correlation between the behavioral improvement and TH-positive cell number in the low-density (1000 cells) transplant group, showing that the hUCMS cells may play a role in rescuing damaged host dopaminergic neurons and promote improvement of motor deficits in PD-model rats. In summary, hUCMS cells appear to be mesenchymal stem cells that can be harvested in great numbers from a non-controversial, inexhaustible source. Human UCMS cells show therapeutic benefit in PD model rats, but the mechanism by which they promote improvement is presently unknown.