Made available in DSpace on 2014-10-09T12:26:39Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:22Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP; Faculdade de Odontologia, Universidade de Sao Paulo, Sao Paulo

Orientador: Fausto Viterbo / Banca: Alexandre L. R. Oliveira / Banca: Amilton Antunes Barreira / Banca: Carlos Nárcio Nóbrega de Jesus / Banca: José de Anchieta de Castro e Horta Junior / Resumo: A neurorrafia látero-terminal sem lesão do nervo doador representou grande avanço na área de microcirurgia de nervos periféricos. O coto distal do nervo receptor é suturado na face lateral do nervo doador, sem prejudicá-lo. Desde 1994, o FK-506, uma droga imunossupressora, tem sido utilizado para impedir a rejeição do aloenxerto nos transplantes de órgãos e apresentou, em alguns estudos, como ação secundária, efeitos positivos sobre a regeneração nervosa periférica. O objetivo desse trabalho foi estudar se a administração de FK-506 traria benefícios na regeneração do nervo após neurorrafia látero-terminal. Foram utilizados 80 ratos Wistar, divididos em 4 grupos experimentais. Cada rato teve seu nervo fibular esquerdo seccionado e a extremidade distal suturada término-lateralmente ao nervo tibial. Os animais do grupo I, II e III foram submetidos à administração de FK-506 com dose de 1,0, 0,5 e 0,25 mg/kg/dia, respectivamente, durante dois meses, enquanto os animas do grupo Controle (GC) não receberam nenhuma droga. Após dois meses da cirurgia, os ratos foram submetidos ao teste da marcha, estudo eletrofisiológico e análise das fibras musculares e nervosas. O grupo II (GII) apresentou o músculo tibial cranial (MTC) com menor massa (p < 0,05) e maior amplitude (p = 0,019). Os resultados do número total de fibras nervosas foram maiores para GII (p < 0,001). Não houve diferença significativa entre os grupos para os índices funcionais. Com base em nosso modelo experimental, pudemos concluir que a administração de FK-506 na dose de 0,5 mg/Kg/dia diminuiu o ganho de massa corporal e massa do músculo tibial cranial e aumentou a regeneração das fibras nervosas, embora não tenha conseguido alterar a resposta funcional / Abstract: The latero-terminal neurorrhaphy without injury to the donor nerve, represented great advances in microsurgery of peripheral nerves. The receptor nerve distal stump was sutured on the lateral side of the donor nerve, without harming it. Since 1994, FK-506, an immunosupressive drug, has been used to prevent allograft rejection in organ transplants and presented, in some studies, as a secondary action, positive effects on peripheral nerve regeneration. The aim of this work was to investigate whether the administration of FK-506 would benefit nerve regeneration after a latero-terminal neurorrhaphy. We used 80 Wistar rats, divided into four experimental groups. Each rat had its left fibular nerve sectioned and the distal stump sutured to the lateral of the tibial nerve. The animals of group I, II and III were subjected to the administration of FK-506 in an amount of 1.0, 0.5 to 0.25mg/K/day, respectively, for two months, while the aminals of the control group received no drug. Two months after surgery the rats were submitted to the walking test, eletrophysiological study and analysis of muscle and nerve fibers. The group II (GII) showed cranial tibial muscle (MTC) with lower mass (p < 0.05) and higher amplitude (p = 0.019). The results by the total number of nerve fibers was higher for GII (p < 0.001) . There was no significant difference between groups for functional indices. Based on our experimental model, we could conclude that the administration of 0.5 mg/Kg/dia of FK-506 decreased body mass gain and mass of MTC and increased regeneration of nerve fibers, although not able to change the functional response / Doutor

Made available in DSpace on 2014-10-09T12:55:10Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:05:55Z (GMT). No. of bitstreams: 0 / O desenvolvimento de moléculas radiomarcadas com alta especificidade para um órgão ou tumor tem contribuído para a obtenção de um diagnóstico de precisão em medicina nuclear.Um caso particular são os peptídeos radiomarcados para a localização de tumores neuroendócrinos como os derivados sintéticos da somatostatina.Atualmente, o DTPA-octreotideo-111In é o radiofármaco mais utilizado com o propósito de visualizar tumores que expressem receptores para somatostatina. Contudo, o uso do indio-111 como radionuclídeo oferece limitações em relação a sua disponibilidade (produto de ciclotron), suas características físicas como meia-vida (67 horas) e emissor de fótons de média energia (171 keV e 245 keV) que não favorecem a obtenção de imagens tipo SPECT (Single Photon Emission Computed Tomography). As propriedades físicas favoráveis do tecnécio-99m (99mTc) fazem dele o radioisótopo mais adequado para substituir o indio-111 (111In) na marcação desses peptídeos. Este trabalho avaliou a preparação e marcação do reagente liofilizado HYNIC-Tyr3-octreotato (HYNIC-octreotato) com 99mTc, baseado em metodologia descrita na literatura, utilizando tricina e EDDA (ácido etilenodiaminadiacetico) como coligantes. Foram estudados os parâmetros de marcação (tempo de incubação, temperatura, volume e atividade do pertecnetato de sódio) e estabilidade do liofilizado. Adicionalmente, estudou-se a influência de pré-congelamento com nitrogênio (N2) líquido na estabilidade do liofilizado, bem como a influência de manitol na pureza radioquímica e biodistribuição do complexo. Os estudos de estabilidade revelaram que o método de liofilização utilizado, empregando o pré-congelamento com nitrogênio líquido possibilitou a obtenção de um reagente liofilizado com estabilidade de 4 meses quando armazenado sob refrigeração. A estabilidade do reagente liofilizado obtido sem pré-congelamento com nitrogênio líquido foi semelhante à obtida com o pré-congelamento.Os estudos de marcações determinaram as melhores condições de marcação, para as quais se obteve pureza radioquímica maior que 90%.A presença de manitol na formulação não influenciou na formação do complexo HYNIC-Octreotato-99mTc, conforme avaliação realizada por CLAE e estudos cintilográficos de distribuição do composto em coelhos.Estudos de biodistribuição invasivos realizados em camundongos Nude com tumor (células AR42J de tumor pancreático) e camundongos Swiss normais, bem como estudos cintilográficos realizados em coelhos e camundongos Nude revelaram cinética de distribuição rápida, acúmulo renal e captação tumoral significativa do peptídeo radiomarcado. Os resultados dos estudos de marcação com 99mTc, produção de reagente liofilizado e biodistribuição sugerem que o radiofármaco HYNIC-octreotato-99mTc apresenta potencial para aplicação em diagnóstico de tumores neuroendócrinos em medicina nuclear. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:26:39Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:22Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP; Faculdade de Odontologia, Universidade de Sao Paulo, Sao Paulo

Abstract The purpose of the present study was to find reliable non-invasive methods for imaging salivary glands and diagnosing primary Sj?gren's syndrome (SS) and to evaluate autonomic function and central nervous system (CNS) disorders in patients with primary SS. The patient population consisted of consecutive patients with primary SS, who fulfilled the International classification criteria for primary SS, from the Division of Rheumatology, Department of Internal Medicine in Oulu University Hospital. Magnetic resonance (MR) imaging and MR sialography of parotid glands were performed on 27 patients and 7 healthy controls and ultrasonography (US) of major salivary glands on 27 patients, 27 healthy controls, and 27 symptomatic controls with sicca symptoms or salivary gland swellings without SS. MR imaging and US showed heterogeneous parenchyma or adipose degeneration of the gland in 81% and 78% of patients, respectively. MR sialography showed ductal system changes, narrowings and dilatations, or cavities in 96% of patients. One healthy control and 2 symptomatic controls had abnormal findings of parotid or submandibular glands on US. Other controls had normal findings. The diagnostic specificity of US was 94%. Parenchymal structural changes on MR imaging and US were associated with anti-Ro/SSA positivity and weakly with the focus score index, but not with salivary or tear secretion, age, disease duration, or features of systemic activity of the disease, such as hypergammaglobulinemia or systemic complications. A comprehensive package of cardiovascular tests, including 24-hour heart rate variability, baroreflex sensitivity test with phenylephrine, Valsalva manoeuvre, deep breathing tests, and active orthostatic test, were conducted on 30 patients and 30 healthy, age and sex-matched, randomly selected population-based controls. No signs of autonomic dysfunction were found in patients compared to controls in any of the tests. The test results were not associated with saliva or tear secretion, age, disease duration, or clinical features of systemic activity of the disease. A case of severe inflammatory CNS disease associated with primary SS was described, and an investigation of the relevant literature was made. Though inflammatory CNS disease is a possible complication of primary SS, there is no consensus regarding its prevalence or significance in the literature. Diagnostics and treatment are empiric. In conclusion, MR imaging, MR sialography, and US yield such a definitive picture of the glandular changes in primary SS that they are promising alternatives for invasive examinations in the diagnostics of primary SS. Comprehensive cardiovascular tests revealed no signs of autonomic dysfunction in patients with primary SS compared to general population.

Affecting 1 in 68 children, Autism Spectrum Disorder (ASD) is one of the most prevalent cognitive disorders in the global population. Symptoms of ASD, although typically not life-threatening, have a large impact on the social wellbeing of diagnosed individuals. Inflammation in the brain, or neuroinflammation, has previously been shown to increase the severity of the behavioral deficits associated with ASD. The exact etiology of the neuroinflammation observed in ASD remains unclear, especially in regards to protein expression that initiates the inflammatory pathway. This experiment examines two specific markers of neuroinflammation, glial fibrillary acidic protein (GFAP) and myelin-associated glycoprotein (MAG) in a previously characterized mouse model of ASD. GFAP is astrocyte-specific, cytoplasmic, and has been shown to be upregulated in trauma or disease pathologies in the brain. MAG is found in the membrane of oligodendrocytes and is a major regulator of development and regeneration of nervous tissue. Control C57bl/6j mice and ASD-representative BTBR T+tf/J (BTBR) mice were sacrificed twenty-one days after birth. Immunoblotting was performed on cingulate cortical tissue using anti-GFAP and anti-MAG primary antibodies to quantify levels of GFAP and MAG protein expression between the control and ASD models. These findings provide further evidence that changes in GFAP and MAG expression may alter the neuroinflammatory pathways observed in ASD-representative mice.

The theme of this dissertation focuses on developing statistical models to learn progression dynamics and mechanisms of neuropsychiatric disorders using data from various domains. Due to limited knowledge about the underlying pathological processes in neurological disorders, it remains a challenge to establish reliable diagnostic criteria and predict disease prognosis in the presence of substantial phenotypic heterogeneity. As a result, current diagnosis and treatment of neurological disorders often rely on late-stage clinical symptoms, which poses barriers for developing effective interventions at the premanifest stage. It is crucial to characterize the temporal disease progression course and study the underlying mechanisms using clinical assessments, blood biomarkers, and neuroimaging biomarkers to evaluate disease stages, identify markers that are useful for early clinical diagnosis, compare or monitor treatment effects and accelerate drug discovery. We propose three projects to tackle challenges in leveraging multi-domain biomarkers and clinical symptoms to learn disease dynamics and progression of neurological disorders: (1) A nonlinear mixture model with subject-specific random inflection points to jointly fit multiple longitudinal markers and estimate marker progression trajectories in a single modality; (2) A multi-layer exponential family factor model integrating multi-domain data to learn lower-dimensional latent space of disease impairment and fully map disease risk and progression; (3) A latent state space model that jointly analyzes multi-channel EEG signals and learns dynamics of different sources corresponding to brain cortical activities. In addition, motivated by the ongoing COVID-19 pandemic, we propose a parsimonious survival-convolution model to predict daily new cases and estimate the time-varying reproduction numbers to evaluate effects of mitigation strategies. In the first project, we propose a nonlinear mixture model with random time shifts to jointly estimate long-term progression trajectories using multivariate discrete longitudinal outcomes. The model can identify early disease markers, their orders of occurrence, and the rates of impairment. Specifically, a latent binary variable representing disease susceptibility status incorporates subject covariates (e.g., biological measures) in the mixture model to capture between-subject heterogeneity. Measures of disease impairment for susceptible patients are modeled jointly under the exponential family framework. Our model allows for subject-specific and marker-specific inflection points associated with patients' characteristics (e.g., genetic mutation) to indicate a critical time when the fastest degeneration occurs. Furthermore, it uses subject-specific latent scores shared among markers to improve efficiency. The model is estimated using an EM algorithm. Extensive simulation studies are conducted to demonstrate validity of the proposed method and algorithm. Lastly, we apply our method to the Parkinson's Progression Markers Initiative (PPMI), and show utility to identify early disease signs and compare clinical symptomatology for the genetic form of Parkinson's Disease (PD) and idiopathic PD. In the second project, we tackle challenges to leverage multi-domain markers to learn early disease progression of neurological disorders. We propose to integrate heterogeneous types of measures from multiple domains (e.g., discrete clinical symptoms, ordinal cognitive markers, continuous neuroimaging and blood biomarkers) using a hierarchical Multi-layer Exponential Family Factor (MEFF) model, where the observations follow exponential family distributions with lower-dimensional latent factors. The latent factors are decomposed into shared factors across multiple domains and domain-specific factors, where the shared factors provide robust information to perform behavioral phenotyping and partition patients into clinically meaningful and biologically homogeneous subgroups. Domain-specific factors capture the remaining unique variations for each domain. The MEFF model also captures the nonlinear trajectory of disease progression and order critical events of neurodegeneration measured by each marker. To overcome computational challenges, we fit our model by approximate inference techniques for large-scale data. We apply the developed method to Parkinson's Progression Markers Initiative (PPMI) data to integrate biological, clinical and cognitive markers arising from heterogeneous distributions. The model learns lower-dimensional representations of Parkinson's disease and the temporal ordering of the neurodegeneration of PD. In the third project, we propose methods that can be used to analyze multi-channel electroencephalogram (EEG) signals intensively measured at a high temporal resolution. Modern neuroimaging technologies have substantially advanced the measurement of brain activities. EEG as a non-invasive neuroimaging technique measures changes in electrical voltage on the scalp induced by cortical activities. With its high temporal resolution, EEG has emerged as an increasingly useful tool to study brain connectivity. Challenges with modeling EEG signals of complex brain activities include interactions among unknown sources, low signal-to-noise ratio and substantial between-subject heterogeneity. In this work, we propose a state space model that jointly analyzes multi-channel EEG signals and learns dynamics of different sources corresponding to brain cortical activities. Our model borrows strength from spatially correlated measurements and uses low-dimensional latent sources to explain all observed channels. The model can account for patient heterogeneity and quantify the effect of a subject's covariates on the latent space. The EM algorithm, Kalman filtering, and bootstrap resampling are used to fit the state space model and provide comparisons between patient diagnostic groups. We apply the developed approach to a case-control study of alcoholism and reveal significant attenuation of brain activities in response to visual stimuli in alcoholic subjects compared to healthy controls. Lastly, motivated by the ongoing COVID-19 pandemic, we propose a robust and parsimonious survival-convolution model aiming to predict COVID-19 disease course and compare effectiveness of mitigation measures across countries to inform policy decision making. We account for transmission during a pre-symptomatic incubation period and use a time-varying effective reproduction number to reflect the temporal trend of transmission and change in response to a public health intervention. We estimate the intervention effect on reducing the infection rate using a natural experiment design and quantify uncertainty by permutation. In China and South Korea, we predicted the entire disease epidemic using only early phase data (two to three weeks after the outbreak). A fast rate of decline in reproduction number was observed and adopting mitigation strategies early in the epidemic was effective in reducing the infection rate in these two countries. The nationwide lockdown in Italy did not accelerate the speed at which the infection rate decreases. In the United States, the reproduction number significantly decreased during a 2-week period after the declaration of national emergency, but declines at a much slower rate afterwards. If the trend continues after May 1, COVID-19 may be controlled by late July. However, a loss of temporal effect (e.g., due to relaxing mitigation measures after May 1) could lead to a long delay in controlling the epidemic.

Amyotrophic lateral sclerosis (ALS) is an incurable and fatal neurodegenerative disease characterized by motor neuron loss. Although pathological mutations exist in >15 genes, the mechanism(s) underlying ALS are unknown. FUS is one such gene and encodes the nuclear RNA-binding protein (RBP), fused in sarcoma (FUS), which actively shuttles between the nucleus and cytoplasm. Intriguingly, nearly half of the ALS mutations identified in FUS cause this protein to mislocalize, suggesting that FUS localization is relevant to disease. Here, we found that excitotoxicity, a neuronal stress caused by aberrant glutamate signaling, induces the rapid redistribution of FUS and additional disease-linked RBPs from the nucleus to the cytoplasm. As excitotoxicity is pathologically associated with ALS, it was notable that the nuclear egress of FUS was particularly robust. Further, ALS-FUS variants that predominantly localize to the nucleus also undergo redistribution. Thus, we sought to understand the purpose underlying FUS translocation and the potential relevance of this response to disease. As calcium dysregulation is strongly associated with neurodegenerative disorders, we examined the contribution of calcium to FUS egress. In addition to global changes to nucleocytoplasmic transport following excitotoxic insult, we observed that FUS translocation caused by excitotoxicity is calcium mediated. Moreover, we found that dendritic expression of Gria2, a transcript encoding an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit responsible for regulating calcium permeability, is FUS-dependent under conditions of stress. Together, these observations support the premise that FUS has a normal function during excitotoxic stress and that glutamatergic signaling may be dysregulated in FUS-mediated ALS.

N-acetylaspartate (NAA) is one of the most abundant molecules in the mammalian central nervous system (CNS). The current paradigm suggests that NAA is synthesized in neurons by the enzyme N-acetyltransferase 8-like (NAT8L) and hydrolyzed into aspartate and acetate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. Although the function of NAA is not well understood, several hypotheses have been proposed since its discovery several decades ago. Among the most cited theory is the concept of acetate delivery to oligodendrocytes via NAA for the synthesis of fatty acids for myelin lipids and myelination. Another concept suggests that NAA functions as a molecular water pump to remove molecular water from oxidative phosphorylation. In contrast, disruption of NAA metabolism has been associated with oxidative stress contributing to neurodegeneration, as seen in Canavan disease, a monogenic disorder associated with loss-of-function mutations in ASPA. Accumulation of NAA in the CNS and peripheral organs is pathognomonic for Canavan disease (CD) and is used clinically to diagnose this rare disease. Symptoms typically occur within months after birth and primarily manifest in the CNS with spongy degeneration of the white matter. Initially, affected patients present with poor feeding, lack of head control, hydrocephalus; later, they miss developmental milestones and develop seizures. Only supportive treatment is available possibly helping patients to survive past the first couple of years. Gene therapy has been considered early on for the treatment of CD. The first trial in humans demonstrated safety but did not result in symptomatic improvement. In addition to gene therapy for the treatment of CD, NAA has gained increasing interest in neurodegenerative and psychiatric disorders, but also in adipose tissue. Here, we are investigating the function of NAA in the context of ASPA deficiency, aka Canavan disease. We found that impaired NAA metabolism caused by ASPA mutations is characterized by a neurometabolic profile that suggests cellular shift from glucose towards fatty acid metabolism for energy production. Although, we found a similar metabolic signature in asymptomatic mice within days after birth, longitudinal comparison suggest that disease progression leads to fatty acid depletion, which is not present in asymptomatic mice, potentially challenging the concept that NAA-derived acetate is essential for lipid synthesis in the myelinating brain. Using rAAV to determine the reversibility of this metabolic phenotype, we found that early treatment prevents loss of myelin, normalizes the neurometabolic phenotype and keeps Canavan mice asymptomatic; in contrast, later treatment only allows for partial normalization of the neurometabolome, despite adequate ASPA gene delivery by rAAV, independent of ubiquitous or astrocyte-restricted hASPA expression. Furthermore, we found that non-enzymatically active hASPA might play a ubiquitous role in glucose uptake regulation in vivo. Importantly, we identified brain regions with metabolic changes that also correspond to the areas with significant histopathologic alterations. Finally, we confirmed the glycolytic changes in a Canavan disease patient cell line using Seahorse metabolic analyzer, demonstrating the decreased rate of glycolysis for energy production. Overall, our findings reveal a novel metabolic phenomenon in Canavan disease and NAA metabolism that allows to assign a novel function of N-acetylaspartate.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by the death of motor neurons in the spinal cord and brain. ALS is a genetically complex disease; diverse mutations cause motor neuron death by disrupting various interrelated pathways. To date, no therapy targeting a single factor can rescue motor neuron loss, nor is it known how or why sub-populations of motor neurons are particularly vulnerable in disease. Many studies have pointed to the Transforming Growth Factor Beta (TGF-𝝱) signaling superfamily as a modifier of disease in human patients and in animal models. Here, we have used the SOD1G93A model of ALS to investigate if and how TGF-𝝱 signaling in motor neurons changes pathology in these animals. In the first part of this study we characterize canonical TGF-𝝱 activation in motor neurons in SOD1G93A animals compared to controls. We have found that a vulnerable motor neuron subpopulation upregulates TGF-𝝱RII, a receptor necessary and unique to the classical arm of the TGF-𝝱 signaling family, in a disease dependent manner. Despite the upregulation of TGF-𝝱RII in these cells, there is not a corresponding activation of downstream canonical TGF-𝝱 effectors in diseased motor neurons. Through in vivo genetic manipulation we found that TGF-𝝱RII is dispensable in motor neurons, but that ablation of TGF-𝝱RI, a key receptor in multiple arms of the TGF-𝝱 superfamily, decreases motor neuron survival in SOD1G93A animals. To further understand how this manipulation changes TGF-𝝱 activation in motor neurons, we performed iterative indirect immunoflourescence imaging. We have identified that knocking out TGF-𝝱RI from motor neurons disrupts downstream canonical TGF-𝝱 activation in these cells. To identify how TGF-𝝱 signaling changes gene expression in these cells we have used Visium, a spatial RNAseq method, on lumbar spinal cords from these animals We have identified and are currently investigating potential downstream targets of TGF-𝝱 signaling in motor neurons in SOD1G93A animals. These data suggest that motor neurons rely on TGF-𝝱 signaling for survival in disease and that TGF-𝝱 signaling is important to the biology of a known vulnerable population of motor neurons.