<p>Parkinson’s disease (PD) is a complex neurodegenerative movement disorder involving protein misfolding, mitochondrial dysfunction, and oxidative stress. The current dissertation, motivated by a lack of valid biomarkers and sustainable therapies, examined the potential application of a novel target for therapeutics and diagnostics of PD — the multifunctional, heat-shock like protein Catecholamine-Regulated Protein 40 (CRP40). The goal of this program of research was to elucidate further the implications of CRP40 in PD using a variety of molecular biology, bioinformatics, and clinical approaches through integrative collaborations with academia, government, and industry partners to translate scientific findings into real world solutions. Chapters 2 and 3 explored the potential therapeutic use and structure-function relationships of CRP40 through elucidating the smallest functional piece of this protein that was six times smaller, and validating a negative control for these experiments (Heat-Shock Protein 47). These initiatives could eventually lead to a small drug that could cross the blood-brain barrier and be targeted to the specific brain regions affected in PD. Chapter 4 examined the potential mechanisms of CRP40, and suggested that this protein may protect neurons from oxidative stress, maintain energy levels, and mitochondrial homeostasis, with important future implications for a variety of disorders. Finally, Chapter 5 presented compelling evidence for the potential use of CRP40 as a valid biomarker for early detection of PD and monitoring of disease progression. Overall, findings suggest that CRP40 may be a critical target for future breakthroughs in the diagnosis and treatment of PD.</p> / Doctor of Science (PhD)

No / Starting from the premise that people are essentially narrative beings, I argue that the onset of severe mental illness compromises the narrative enterprise of being able to construct one's Self and one's relationships inmeaningful and coherent ways. This is due to both the curtailment of opportunities for narrative engagement and the dispossession of those whose narratives do not conform to the current conceptualization of narrative and narrativity. In these circumstances, supporting the narrative enterprise is an ethical endeavour that requires that we examine not only which narratives we construct, but also how we construct them. This requires a re-thinking of what might constitute narrative and how we might facilitate or enhance the narrativity of people with severe mental illness. Following this, I suggest four means to support the narrativity of people with severe mental illness: through maintaining narrative continuity, maintaining narrative agency, countering master narratives and attention to small stories.

Neurological disorders present a significant challenge in global health. With the increasing availability of imaging datasets and the development of precise machine learning models, early and accurate diagnosis of neurological conditions is a promising and active area of research. However, several characteristic factors in neurology domains, such as heterogeneous imaging, inaccurate labels, or limited data, act as bottlenecks in using deep learning on clinical neuroimaging. Given these circumstances, this dissertation attempts to provide a guideline, proposing several methods and showcasing successful implementations in broad neurological conditions, including epilepsy and neurodegeneration. Methodologically, a particular focus is on comparing a two-dimensional approach as opposed to three-dimensional neural networks. In most clinical domains of neurological disorders, data are scarce and signals are weak, discouraging the use of 3D representation of raw scan data. This dissertation first demonstrates competitive performances with 2D models in tuber segmentation and AD comorbidity detection. Second, the potentials of ensemble learning are explored, further justifying the use of 2D models in the identification of neurodegeneration. Lastly, CleanNeuro is introduced in the context of 2D classification, a novel algorithm for denoising the datasets prior to training. CleanNeuro, on top of 2D classification and ensemble learning, demonstrates the feasibility of accurately classifying patients with comorbid AD and cerebral amyloid angiopathy from AD controls. Methods presented in this dissertation may serve as exemplars in the study of neurological disorders using deep learning and clinical neuroimaging. Clinically, this dissertation contributes to improving automated diagnosis and identification of regional vulnerabilities of several neurological disorders on clinical neuroimaging using deep learning. First, the classification of patients with Alzheimer’s disease from cognitively normal group demonstrates the potentials of using positron emission tomography with tau tracers as a competitive biomarker for precision medicine. Second, the segmentation of tubers in patients with tuberous sclerosis complex proves a successful 2D modeling approach in quantifying neurological burden of a rare yet deadly disease. Third, the detection of comorbid pathologies from patients with Alzheimer’s disease is analyzed and discussed in depth. Based on prior findings that comorbidities of Alzheimer’s disease affect the brain structure in a distinctive pattern, this dissertation proves for the first time the effectiveness of using deep learning on the accurate identification of comorbid pathology in vivo. Leveraging postmortem neuropathology as ground truth labels on top of the proposed methods records competitive performances in comorbidity prediction. Notably, this dissertation discovers that structural magnetic resonance imaging is a reliable biomarker in differentiating the comorbid cereberal amyloid angiopathy from Alzheimer’s disease patients. The dissertation discusses experimental findings on a wide range of neurological disorders, including tuberous sclerosis complex, dementia, and epilepsy. These results contribute to better decision-making on building neural network models for understanding and managing neurological diseases. With the thorough exploration, the dissertation may provide valuable insights that can push forward research in clinical neurology.

Friedreich's ataxia (FRDA) is the most prevalent inherited ataxia, affecting one in every 50,000 individuals in the United States. This hereditary condition is caused by an abnormal GAA trinucleotide repeat expansion within the first intron of the frataxin gene resulting in decreased levels of the frataxin protein (FXN). Insufficient cellular frataxin levels results in iron accumulation, increased reactive oxygen species production and mitochondrial dysfunction. Tissues most heavily impacted are those most dependent on oxidative phosphorylation as an energy source and include the nervous system and muscle tissue. This is evident in the clinical phenotype which includes muscle weakness, ataxia, neurodegeneration and cardiomyopathy. However, there has been a lack of data regarding the cell type specific contributions in FRDA pathogenesis. We generated a cohort of induced pluripotent stem cells (iPSCs) consisting of FRDA patient lines, CRISPR-Cas9 edited controls, carriers and non-related controls. Our preliminary data identified a hyperinflammatory microglial phenotype with extensive defects in mitochondrial function; since microglia are the primary innate immune cell of the brain, we hypothesized microglia may decrease neuronal viability which contributes to FRDA pathology. To investigate this, the iPSC cohort was utilized to generate microglia (iMGs) and neurons to better understand microglia-mediated neurodegeneration and how this contributes to pathology. An in vitro co-culture model composed of neurons, astrocytes and microglia was employed to better understand microglia-neuronal communication in FRDA. Healthy neurons co-cultured with FRDA iMG or with FRDA iMG-conditioned media demonstrated higher incidences of caspase-3 mediated apoptosis. These findings were recapitulated in vivo as xenotransplantation of FRDA microglia progenitors into a murine model resulted in reduced Purkinje cell survival in the cerebellum. Previous research has demonstrated the therapeutic potential of wildtype microglia to rescue the FRDA phenotype in the Y8GR mouse model of FRDA. To further explore the potential mechanisms behind this rescue, the delivery of mitochondria and FXN to FRDA microglia and neurons was investigated. CRISPR-Cas9 edited microglia demonstrated transfer of healthy mitochondria to FRDA microglia and neurons in an in vitro co-culture model. To investigate the transfer of frataxin protein, an FRDA iPSC line was transduced with an FXN-GFP lentivirus. Restoring FXN expression was demonstrated to rescue the FRDA microglial morphological phenotype. FXN-GFP microglia demonstrated transfer of frataxin protein to FRDA microglia suggesting the potential role of microglia as a therapeutic vehicle in FRDA. Together these findings show that FRDA microglia have a deleterious effect on neuronal viability, while healthy microglia may work as a therapeutic vehicle through the delivery of mitochondria and frataxin to FRDA cells.

T cells infiltrate the degenerating brain and influence central nervous system (CNS) inflammation and neuronal health. In mice, the choroid plexus and the meninges have been implicated in regulating T cell entry and egress from the CNS, respectively. Further, antigen presenting cells in the mouse meninges present CNS-derived antigens to T cells and may represent a method for the peripheral immune system to sense and respond to CNS immune triggers. However, whether these processes occur in the human choroid plexus and meninges has not been comprehensively studied. Further, the antigens towards which T cells in the degenerating human brain and its borders respond remain unknown. Therefore, I implemented a multi-omics approach using fresh postmortem tissue from patients diagnosed with amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), and non-neurodegenerative controls to identify not only the T cell-associated changes that occur in the degenerating human CNS and surrounding tissues but also identified a library of putative antigen targets for disease-associated T cell populations. Specifically, using single cell RNA and TCR sequencing information from paired postmortem choroid plexus, leptomeninges, and brain I lineage traced T cells using their TCR information and found that T cell access to leptomeninges and brain is likely limited and controlled by anti-inflammatory macrophage activity at the blood/CSF barrier (BCSFB). Once past the BCSFB, I present evidence that T cells access the CNS where they interact with MHC expressed by microglia. T cells also accumulate in the leptomeninges where they become tissue resident memory T cells. These tissue resident memory T cells likely serve as a reservoir for a rapid antigen-driven immune response to future CNS inflammatory insults. Finally, by performing immunopeptidomics to identify peptides presented by MHC in the same patients’ CNS and border tissues, I identified a library of putative antigenic triggers that may drive high levels of T cell clonal expansion in the brain and surrounding tissues. Altogether, this thesis serves as a resource for understanding the trajectory of T cells as they travel into the degenerating human brain and as a foundation for the development of antigen-specific precision medicines to treat neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease affecting mostly spinal motor neurons. Its hallmarks include motor neuron loss, muscle atrophy, paralysis and death. Studies in ALS using genetic mutants in mouse models have identified numerous changes in motor neurons at early stages, including excitotoxicity, oxidative stress, denervation at neuromuscular junction (NMJ), mitochondrial abnormalities and motor neuron excitability. Such changes are accompanied by abnormal motor unit function and motor output. Despite attempts to identify early changes in ALS, the causal relationship between abnormal motor output – broadly defined as motor unit dysfunction – and the reported pathological changes are poorly understood. Here, we used C57BL6/J-SOD1 G93A male mice as the animal model of ALS, together with behavioral, physiological and morphological assays to identify transcriptional changes in vulnerable motor neurons involved in motor dysfunction during ALS onset. We found that ALS mice travelled shorter distances as early as one month old, which progressively reduced to peak around ~P80 and dramatically decreased afterwards until death at ~P150. To identify changes responsible for the observed reduction in distance travelled in SOD1 mutants, we investigated mice at ages in which behavioral differences became apparent. We opted to evaluate the extent of motor unit pathology by focusing on a motor pool involved in running that is known to be vulnerable in ALS, the tibialis anterior (TA) muscle. Although TA motor units do not exhibit any detectable overt changes between SOD1 and control mice at P30, SOD1-G93A mice at P50 exhibited a significant ~40% loss of functional TA motor units, determined by in vivo experiments in which individual motor units were counted following incremental stimulation intensities and quantifying the elicited muscle force. This reduction was not accompanied by motor neuron loss. However, the loss in functional motor units was accompanied by significant decrease in the twitch force alongside ~40% NMJ denervation in the TA muscle. Taken together, the functional changes in motor units and NMJ denervation are deficits in motor unit function that can account for the reduction in distance travelled daily by SOD1 male mice. We next wanted to identify transcriptional changes in the TA motor neurons of SOD1-G93A mice that could be involved in the observed motor unit pathology. To this end, large TA motor neurons (presumed to be motor neurons from the affected vulnerable motor units) from control and SOD1 males at P30 and P50 were subjected to RNAseq which were selectively collected via laser capture microdissection following labelling with a fluorescent tracer through intramuscular injection. The resultant differentially expressed genes included 6 upregulated and 10 downregulated transcripts. Among these, a mitochondrial gene, Chchd10, which mutations have recently been associated with ALS, revealed elevated levels in motor neurons. Reduction of Chchd10 protein levels within CNS, including motor neurons, via intracerebroventricular injection with an antisense oligonucleotide, we observed a significant protection against motor unit loss that was translated with an increase in distance travelled daily by ALS-treated mice. These results suggest that dysregulation of Chchd10 in SOD1 G93A is involved in the pathogenesis of disease. Furthermore, whilst the downregulation of Chchd10 did not rescue all characteristics of motor unit pathology it implies that multiple genes dysregulation may be involved and be responsible for the different characteristics of motor unit pathology early in the disease onset. It further highlights the downregulation of Chchd10 as a potential novel therapeutic target for ALS.

This work seeks to contribute to the discipline of neuropharmacology by way of structure activity relationship from the standpoint of an organic chemist. More specifically, we sought to develop robust synthetic methodology able to efficiently produce an array of compounds for the purpose of systematic evaluation of their interaction with specific sights within the central nervous system (CNS) in order to better understand the mind and to develop drugs that may have beneficial effects on neurological function. The focus of these studies has been toward the development of novel molecules, using a structure-activity relationship approach, that exhibit binding affinity at specific targets within the CNS. The merit of such studies is twofold: primarily, new compounds are produced that provide valuable scientific insight about their physiological targets, and secondarily, new synthetic methodologies that may arise in order to produce these compounds, thereby contributing to the whole of organic chemistry. As a result of the research described herein, the development of one high affinity and several moderate affinity compounds at the cannabinoid receptor subtype 1 (CB1) has been accomplished. The research demonstrates that a diaryl ether molecular scaffold represents a successful motif in the cannabinoid pharmacophore. The production of the compounds in the SAR studies also introduced a novel general synthetic methodology for the synthesis of diaryl ethers around a phloroglucinol core. A second project was initiated in order to explore the synthetic methods required to develop a general process for the synthesis of rigid aminobenzocyclobutane analogs of known phenethylamines with activity at monoaminergic neurotransmitter sites. Using the synthetic approach devised here, four novel aminobenzocyclobutane isomeric analogs of a known pharmacologically active phenethylamine, (RS)-phenylpropan-amine were synthesized and are currently being evaluated for pharmacological potential.

Introdução - Alterações cardíacas na anemia falciforme (AF) são frequentes e iniciam-se precocemente. Há evidências de que exista também disfunção na regulação do sistema nervoso autônomo o que pode contribuir com eventos de morbidade. Objetivos Avaliar a modulação autonômica cardíaca por meio da variabilidade da frequência cardíaca em crianças e adolescentes com anemia falciforme. Método - Estudo analítico no qual foi realizada uma comparação da variabilidade da frequência cardíaca em 45 crianças e adolescentes, menores de 20 anos, com anemia falciforme, com um grupo controle pareado um a um por idade e sexo. A frequência cardíaca foi obtida pelo frequencímetro de pulso e analisada, batimento a batimento. Estes pacientes são usuários do ambulatório de hematologia pediátrica do Sistema Único de Saúde. Esta pesquisa está em consonância com a resolução 466/2012 do Ministério da Saúde. Resultados - Observamos diferença significativa nos índices do domínio da frequência (VLF, LF, HF e LF/HF). Estas diferenças não foram observadas nos pacientes em uso de hidroxiureia. Conclusão - Existe uma disfunção autonômica na AF que ocorre desde a infância, podendo estar relacionada a uma menor modulação do simpático e uma maior modulação do parassimpático. Esta diferença não foi observada em pacientes em uso de hidroxiureia / Introduction - Cardiac changes in sickle cell disease (AF) are frequent and begin early. There is evidence that there is also dysfunction in the regulation of the autonomic nervous system, which may contribute to morbidity events. Objectives - To evaluate the autonomic cardiac modulation by heart rate variability in children and adolescents with sickle cell anemia. Method - An analytical study comparing the heart rate variability of 45 children and adolescents, younger than 20 years, with sickle cell anemia, with a control group matched one by one by age and sex. The heart rate was obtained by pulse frequency and analyzed, beat by beat. These patients are attending the pediatric hematology outpatient of the National Health System. Results - We observed a significant difference in the frequency domain indexes (VLF, LF, HF and LF / HF). The results of this study are in agreement with resolution 466/2012 of the Ministry of Health of Brazil. These differences were not observed in patients taking hydroxyurea. Conclusion - There is an autonomic dysfunction in AF that occurs from childhood, and may be related to a lower modulation of the sympathetic and greater modulation of the parasympathetic. This difference was not observed in patients taking hydroxyurea

Foi caracterizada uma amostra de 81 doentes com dor relevante crônica pélvica de origem indefinida e na ausência de afecções viscerais, segundo a expressão sintomática, aspectos clínicos e síndromes álgicas. A média das idades foi de 48,6 anos. Foram identificadas anormalidades miofasciais pélvica em 55 doentes (67,9%), neuropáticas em 10 (12,3%) e miofasciais e neuropáticas em 16 (19,8%). A descrição verbal da dor e as regiões acometidas pela dor, impactos funcionais, a evidenciação de pontos dolorosos e em gatilhos e os aspectos psicossociais dos doentes foram estabelecidos para melhorar a compreensão da condição clínica dos doentes. Traumatismos decorrentes de traumatismos cirúrgicos estavam relacionados à ocorrência da dor em 36 (44,4%) doentes, sendo dor miofascial em 15 doentes (18,5%), 6 (7,4%) como neuropática e 15 (18,5%) como dor miofascial e neuropática / Eighty-one chronic pelvic pain patients (mean of ages 48.6 years old) without visceral abnormalities and uncertain etiology were characterized according to the symptomatic expression, clinical findings and pain syndromes. It was observed that in 55 patients there was pelvic miofascial pain (67.9%), in 10 patients neuropathic (12.3%) conditions and 16 (19.8%) had miofascial and neuropathic abnormalities. The verbal description of pain, pain aspects as localization, aggravating and improvement factors, functional impact, the occurrence of trigger or tender points and the psychosocial aspects were determined to improve the comprehension of the clinical presentation of the patients. Surgical traumas were related to pain in 26 (32.0%) patients, miofascial pain in 11 (13.6%), neuropathic pain in 5 (6.2%) and miofascial and neuropathic pain in 10 (12.3%)

INTRODUÇÃO: A neuropatia periférica induzida por quimioterapia (NPIQ) está entre os efeitos colaterais mais comuns decorrentes da quimioterapia antineoplásica e uma das principais causas da redução da dose ou interrupção do tratamento. Os sintomas mais prevalentes são a dor e a parestesia, acarretando desconfortos crônicos e perda de habilidades funcionais, interferindo negativamente na qualidade de vida dos pacientes. Estudos recentes têm avaliado o uso da Estimulação Elétrica Nervosa Transcutânea (TENS) nesta patologia, apresentando evidências positivas na redução da dor, porém seu efeito nos sintomas de parestesia e nas atividades de vida diária destes pacientes ainda não foram avaliados. OBJETIVO: Investigar os efeitos da Estimulação Elétrica Nervosa Transcutânea (TENS) nos sintomas de dor, parestesia e nas atividades de vida diária da NPIQ em indivíduos com diagnóstico de câncer de mama e colorretal, submetidos ao tratamento de quimioterapia. MÉTODOS: Trata-se de um ensaio clínico duplo-cego, controlado, randomizado e multicêntrico, com abordagem quantitativa, em pacientes submetidos ao tratamento de quimioterapia, contendo em seu protocolo os seguintes quimioterápicos: paclitaxel e oxaliplatina. Os sujeitos da pesquisa utilizaram o dispositivo terapêutico TENS com modulação de frequência entre 7 e 75 Hz na região distal dos membros, no local de maior desconforto, com intervenções diárias de 60 minutos, durante três ciclos de quimioterapia (45 dias). Os participantes foram divididos em dois grupos: grupo TENS ativa (GTA) e grupo TENS placebo (GTP). A avaliação dos efeitos da TENS foi medida através dos seguintes instrumentos: a Escala Visual Analógica (EVA) para avaliar os sintomas de dor e parestesia e Questionário de Neurotoxicidade Induzida por Anti-neoplásicos (QNIA) para avaliação dos sintomas da NPIQ. RESULTADOS: Finalizaram a pesquisa 24 pacientes. Não se observou uma diferença significativa entre os 2 grupos no que se refere ao desfecho primário de redução dos sintomas de dor (p = 0.666), parestesia (p = 0,673) e impacto da TENS na frequência dos sintomas (p = 0,5906) e atividades de vida diária (p = 0,8565). CONCLUSÃO: Estes resultados sugerem que a TENS aplicada no modo de modulação de frequência não foi eficaz para melhorar os sintomas de neuropatia periférica induzida por quimioterapia, durante os ciclos de quimioterapia. Não houve, porém, agravamento dos sintomas em ciclos subsequentes ao início dos sintomas da doença / BACKGROUND: Peripheral neuropathy induced by chemotherapy (PNIC) is amongst the most common side effects derived from antineoplastic chemotherapy and one of the principal causes of dose reduction or treatment interruption. The most prevalent symptoms are pain and numbness, resulting from chronic discomfort to loss of functional abilities, negatively affecting quality of life and autonomy of patients. Recent studies have evaluated the use of Transcutaneous Electrical Nerve Stimulation (TENS) in this disease, pointing to evidence of pain reduction, but its effect on symptoms of paresthesia and in daily life activities have not yet been evaluated. OBJECTIVE: To investigate the effects of Transcutaneous Electrical Nerve Stimulation (TENS) for reducing the symptoms of pain, paresthesia and the daily activities of PNIC in patients diagnosed with breast cancer and colorectal cancer undergoing chemotherapy treatment. METHODS: It is a double-blind, controlled, randomized, multicenter clinical trial with a quantitative approach in a sample of 24 patients undergoing chemotherapy treatment, containing in its protocol the following chemotherapeutic agents: paclitaxel and oxaliplatin. The research subjects used the TENS therapeutic device with frequency modulation between 7 and 75 Hz in the distal limb, on the location of greatest discomfort with daily interventions lasting 60 minutes for three chemotherapy cycles (45 days). Participants were divided into two groups: active TENS group (ATG) and placebo TENS group (PTG). The assessment of the effects of TENS was measured by the following instruments: The Visual Analogue Scale (VAS) to assess the symptoms of pain and numbness and Questionnaire for Neurotoxicity Induced by Anti-neoplastic (QNIA) to assess the symptoms of PNIC. RESULTS: A 24-patient study was completed. There was no significant difference between the two groups regarding the primary endpoint of reduced pain symptoms (p = 0.666) and paresthesia (p = 0.673), neither any measurable impact of TENS in the frequency of symptoms (p = 0.5906) or activities of daily living (p = 0.8565). CONCLUSION: These results suggest that TENS applied in frequency modulation mode is not effective for ameliorating the symptoms of peripheral neuropathy induced by chemotherapy during chemotherapy cycles. There was, however, no worsening of symptoms in subsequent cycles after the onset of symptoms