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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.

Made available in DSpace on 2017-07-24T19:38:08Z (GMT). No. of bitstreams: 1 Taissa Adriana Costin.pdf: 4929780 bytes, checksum: 1674ff97dbc8df45d3aedf8e6744fdde (MD5) Previous issue date: 2013-03-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Due to the great importance of the aspartic acid, since it is found in the Dimethyl N-acetylaspartate in high concentrations in the urine of patients with different neurological problems, an example is Canavan disease, which has been associated with elevated concentrations of N-acetylaspartate in cerebrospinal fluid, it inspired the interest of studying conformational equilibrium of compounds with structures similar to Dimethyl N-Acetylaspartate. The focus of this study was to research the compound structural Dimethyl N-acetylaspartate (NAA) and their derivatives Dimethyl N-Methylaspartate (NMA) and Dimethyl N, N- Dimethylaspartate (NDA). The principle was to establish the most stable geometries and populations of conformers involved in the equilibrium phase and isolated in solution. To perform DFT calculations, we used the Gaussian 03 software package. The potential energy surfaces (SEPs) were obtained with B3LYP/6-311G level of theory and the points of lowest energy, made possible as more stable geometries which were optimized with the level of theory B3LYP/cc-PVTZ. For the structure of the NAA, we found seven more stable geometries. The majority conformer of NAA showed a population of 31,9% on balance. For the structure of the NMA met ten most stable geometries. The majority conformer population showed a 35,4% on the balance. As for the structure of the NDA, it also met seven most stable geometries. The majority conformer population showed a 46,5% on the balance. Besides the equilibrium phase alone was evaluated with these structures and solvation calculations it was observed that populations of conformers were changed, but the same major conformer being isolated majority remained in solution. In order to compare the theoretical data with experimental data, these compounds were synthesized therefore performed NMR spectroscopic analysis which analyzed the coupling constants 2J and 3J and IV, held the deconvolution of the carbonyl band. / Devido à grande importância do ácido aspártico, já que o mesmo é encontrado na forma de N-Acetilaspartato em concentrações elevadas na urina de pacientes com diferentes problemas neurológicos, um exemplo é a doença de Canavan, a qual vem sendo associada a concentrações elevadas de N- Acetilaspartato no fluido cerebroespinal despertou-se o interesse em estudar o equilíbrio conformacional de compostos com estruturas semelhantes ao mesmo. O foco deste estudo foi realizar uma investigação estrutural do composto N-Acetilaspartato de dimetila (NAA) e dos seus derivados, N-Metilaspartato de dimetila (NMA) e N,N-Dimetilaspartato de dimetila (NDA), determinando as geometrias mais estáveis e as populações dos confôrmeros envolvidos no equilíbrio na fase isolada e em solução. Para a realização dos cálculos DFT, utilizou-se o pacote de programas Gaussian 03. As superfícies de energia potencial (SEPs) foram obtidas com nível de teoria HF/6-311G e os pontos de menor energia, assumidos como possíveis geometrias mais estáveis, as quais foram otimizadas com o nível de teoria B3LYP/cc-PVTZ. Para a estrutura do NAA, encontrou-se sete geometrias mais estáveis. O confôrmero majoritário do NAA apresentou uma população relativa de 31,9% do equilíbrio. Para a estrutura do NMA, encontrou-se dez geometrias. O confôrmero majoritário apresentou uma população relativa a 35,4% do equilíbrio. No caso da estrutura do NDA, obteve-se também sete geometrias. O confôrmero majoritário apresentou uma população relativa a 46,5% do equilíbrio. Além do equilíbrio em fase isolada, avaliou-se estas estruturas com cálculos de solvatação e observou-se que as populações dos confôrmeros foram alteradas, porém os mesmos confôrmeros majoritário em fase isolada, continuaram majoritários em solução. A fim de confrontar os dados teóricos com dados obtidos experimentalmente, estes compostos foram sintetizados e, portanto realizadas análises espectroscópicas de RMN a qual analisou-se as constantes de acoplamento 2J e 3J e no IV, realizou-se a deconvolução da banda da carbonila.

Orientador: Gabriela Castellano / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-21T20:54:35Z (GMT). No. of bitstreams: 1 Landim_RicardoCesarGiorgetti_M.pdf: 17116375 bytes, checksum: 1ddc2a2b2cc8da480f9f40a006e4144a (MD5) Previous issue date: 2013 / Resumo: As variações metabólicas que ocorrem no cérebro subjacentes á ativação neuronal ainda estão longe de ser bem compreendidas e, portanto, constituem objeto de estudo de grande interesse por parte da comunidade cientifica da área. Uma forma de estudar estas variações é por meio da técnica de espectroscopia funcional por ressonância magnética (functional Magnetic Resonance Spectroscopy - fMRS), na qual espectros de uma dada região cerebral são coletados de forma dinâmica, enquanto o indivíduo é sujeito a algum estimulo sensorial ou tarefa cognitiva. Em particular, o N-acetil-aspartato (NAA) _e o metabólito que gera o principal pico encontrado em espectros cerebrais de espectroscopia por ressonância magnética utilizando o núcleo do hidrogênio. Na verdade, este pico provém do próprio NAA (90%) e também de um derivado deste, o N-acetil-aspartil-glutamato (NAAG). Em experimentos de fMRS realizados por nosso grupo, foi encontrada variação de NAA e NAAG no córtex visual de indivíduos normais, quando estes são sujeitos á apresentação de um estimulo visual. Porem, as variações do sinal de MRS do NAA em experimentos de fMRS são um assunto controverso na literatura da área, pois há trabalhos que apontam para sua variação com o estímulo e outros que dizem que não há variações. Além disso, os trabalhos existentes na literatura mediram a variação conjunta de NAA e NAAG. Dessa forma, para elucidar o que acontece com estes metabólitos durante a ativação cerebral seria interessante medí-los separadamente. O objetivo principal deste trabalho foi, portanto, realizar experimentos de fMRS nos quais estes metabólitos pudessem ser medidos de forma independente, através da implementação da sequência de pulsos MEGA-PRESS, que separa as contribuições do NAA e NAAG já no momento da medida. Os experimentos foram realizados em sujeitos saudáveis durante estimulação visual. Foram realizados vários testes para se padronizar o pré -processamento e duas metodologias foram utilizadas. Diversas quantificações foram feitas e como resultado foram encontradas variações de NAA (diminuição) e NAAG (aumento) durante o estímulo. Os resultados concordam com os apresentados em dois dos três trabalhos similares encontrados na literatura, e também com os resultados anteriores de nosso grupo. A diminuição de NAA pode ser explicada através da hipótese de que ele funcione como uma bomba de _agua molecular, enquanto que a produção de NAAG concorda com o fato de que este metabólito _e um neuropeptídeo que é liberado nas sinapses e atua como modulador para a liberação de certos neurotransmissores, e também concorda com modelos para o metabolismo energético que apontam para este metabólito como relacionado á resposta hiperêmica vascular que origina o sinal BOLD / Abstract: Metabolic changes that occur in the brain underlying neuronal activation are still far form being understood and, therefore, are a subject matter of great interest for the scientific community of this research field. One way to study these variations is through the technique of functional Magnetic Resonance Spectroscopy (fMRS), in which spectra from a given brain region are collected dynamically, while the volunteer is subject to some sensory stimulus or cognitive task. In particular, N-acetyl-aspartate (NAA) is the metabolite that generates the main peak found the MR spectra of the brain using the hydrogen nucleus. Indeed, this peak originates from NAA itself (90 %) and also from a derivative thereof, N-acetylaspartyl-glutamate (NAAG). In fMRS experiments performed by our group, NAA and NAAG changes were found in the visual cortex of normal individuals when they are subject to a visual stimulus. However, the MRS signal variations of NAA in fMRS experiments are a controversial subject in the literature, since there are studies that point to its variation with the stimulus and others that show that there is no variation. In addition, the existing works measured the joint NAA and NAAG variation. Thus, to elucidate what happens to these metabolites during brain activation it would be interesting to measure them separately. The main objective of this study was, therefore, to perform fMRS experiments in which these metabolites could be measured independently by implementing the MEGA-PRESS pulse sequence, which separates the NAA and NAAG contributions at the time of measurement. The experiments were performed in healthy subjects during visual stimulation. Several tests were performed to standardize the preprocessing and two methods were used. Several quantifications were made and as result NAA and NAAG variations (decrease and increase, respectively) were found during the stimulus. The results agree with those presented in two out of three similar studies found in the literature, and also with the previous results from our group. The NAA decrease may be explained through the hypothesis that it works as a molecular water pump, whereas the NAAG production agrees with the fact that this metabolite is a neuropeptide that is released at the synapses and acts as a modulator for the release of certain neurotransmitters, and also agrees with models for energy metabolism that point to this metabolite as related to the vascular hyperemic response that originates the BOLD signal / Mestrado / Física / Mestre em Física

Proton MR spectroscopy is a non-invasive tool for measuring in vivo concentrations of several metabolites. The aim of this thesis was to test its applicability and reliability in neurosurgical praxis. In the first part of the study multiple MR spectroscopy methods were applied in a group of patients after surgery and oncologic treatment for high-grade glioma to test which method performed best in discriminating recurrent tumor from radionecrosis in the presence of a new enhancing lesion. The best diagnostic yield was achieved by comparison of choline, creatine and lactate between lesion and contralateral side (sensitivity 93.3%, specificity 78.6%). Creatine was significantly decreased in patients compared to controls. The inhibiting effect of ongoing oncologic treatment on cerebral and tumoral metabolism makes differential diagnosis trickier. Therefore, a diagnosis of radionecrosis assessed during ongoing radio- and chemotherapy should be confirmed after its completion. In the second part of the study MR spectroscopy data was compared with MR hippocampal volumetry and transcranial doppler examination in a cohort of patients with unilateral occlusion of the internal carotid artery. The N-acetylaspartate/choline ratio and hippocampal volume were significantly lower in both hemispheres of patients...

<div> Advancements in cancer treatments have increased rapidly in recent years, but cures remain elusive. Surgical tumor resection is a central treatment for many solid malignancies. Residual tumor at surgical margins leads to tumor recurrence. Novel tools for assessing residual tumor at surgical margins could improve surgical outcomes by helping to maximize the extent of resection. Ambient ionization-mass spectrometry (MS) methods generate and analyze ions from minimally prepared samples in near-real-time (e.g. seconds to minutes). These methods leverage the high sensitivity and specificity of mass spectrometry for analyzing gas phase ions and generating those ions quickly and with minimal sample preparation. Recent work has shown that differential profiles of ions, corresponding to phospholipids and small metabolites, are detected from cancerous and their respective normal tissue with ambient ionization-MS methods. When properly implemented, ambient ionization-MS could be used to assess for tumor at surgical margins and provide a molecular diagnosis during surgery. </div><div><br></div><div>The research herein reports efforts in developing rapid intraoperative ambient ionization-MS methods for the molecular assessment of cancerous tissues. Touch spray (TS) ionization and desorption electrospray ionization (DESI) were utilized to analyze kidney cancer and brain cancer.</div><div><br></div><div> As a demonstration of the applicability of TS-MS to provide diagnostic information from fresh surgical tissues, TS-MS was used to rapidly analyze renal cell carcinoma and healthy renal tissue biopsies obtained from human subjects undergoing nephrectomy surgery. Differential phospholipid profiles were identified using principal component analysis (PCA), and the significant ions were characterized using multiple stages of mass spectrometry and high resolution/exact mass MS. The same TS-MS analyzed renal tissues were subsequently analyzed with DESI-MS imaging to corroborate the TS-MS results, and the significant DESI-MS ions were also characterized with MS.</div><div><br></div><div>Significant efforts were made in developing and evaluating a standalone intraoperative DESI-MS system for analyzing brain tissue biopsies during brain tumor surgery. The intraoperative DESI-MS system consists of a linear trap quadrupole mass spectrometer placed on a custom-machined cart that contains all hardware for operating the mass spectrometer. This instrument was operated in the neurosurgical suites at Indiana University School of Medicine to rapidly analyze brain tissue biopsies obtained from glioma resection surgeries. A DESI-MS library of normal brain tissue and glioma was used to statistically classify the brain tissue biopsies collected in the operating room. Multivariate statistical methodologies were employed to predict the disease state and tumor cell percentage of the samples. A DESI-MS assay for detecting 2-hydroxyglutarate (2HG), the oncometabolic product of the isocitrate dehydrogenase (IDH) mutation (a key glioma prognostic marker), was developed and applied to determine the IDH mutation status during the surgical resection. The strengths, weaknesses, and areas of future work in this field are discussed. </div><div><br></div>