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The Edwardsiella genus presents five different pathogenic species: Edwardsiella tarda, E. anguillarum, E. piscicida, E. hoshinae and E. ictaluri. These species cause infections mainly in fish, but they can also infect reptiles, birds or humans. Lipopolysaccharide (endotoxin) plays an important role in the pathogenesis of these bacteria. For the first time, the chemical structure and genomics of the lipopolysaccharide (LPS) core oligosaccharides of E. piscicida, E. anguillarum, E. hoshinae and E. ictaluri were studied. The complete gene assignments for all core biosynthesis gene functions were acquired. The structure of core oligosaccharides was investigated by ¹H and 13C nuclear magnetic resonance (NMR) spectroscopy. The structures of E. piscicida and E. anguillarum core oligosaccharides show the presence of →3,4)-L-glycero-α-D-manno-Hepp, two terminal β-D-Glcp, →2,3,7)-L-glycero-α-D-manno-Hepp, →7)-L-glycero-α-D-manno-Hepp, terminal α-D-GlcpN, two →4)-α-D-GalpA, → 3)-α-D-GlcpNAc, terminal β-D-Galp and →5-substituted Kdo. E. hoshinare core oligosaccharide shows only one terminal β-D-Glcp, and instead of terminal β-D-Galp a terminal α-D-GlcpNAc. E. ictaluri core oligosaccharide shows only one terminal β-D-Glcp, one →4)-α-D-GalpA and do not have terminal α-D-GlcpN (see complementary figure).

In this study, half-sandwich Ru(II) complexes containing acylthiourea ligands of the general type [Ru(η6-p-cymene)(PPh3)(S)Cl]PF6 (1m–6m) and [Ru(η6-p-cymene)(PPh3)(S–O)]PF6 (1b–6b) where S/S–O = N...

The 235U(n,f) cross section was measured in a wide energy range (18 meV–170 keV) at the n_TOF facility at CERN, relative to 6Li(n,t) and 10B(n,α) standard reactions, with high resolution and accuracy, with a setup based on a stack of six samples and six silicon detectors placed in the neutron beam. In this paper we report on the results in the region between 18 meV and 10 keV neutron energy. A resonance analysis has been performed up to 200 eV, with the code SAMMY. The resulting fission kernels are compared with the ones extracted on the basis of the resonance parameters of the most recent major evaluated data libraries.Acomparison of the n_TOF datawith the evaluated cross sections is also performed from thermal to 10 keV neutron energy for the energy-averaged cross section in energy groups of suitably chosen width. A good agreement, within 0.5%, is found on average between the new results and the latest evaluated data files ENDF/B-VIII.0 and JEFF-3.3, as well as with respect to the broad group average fission cross section established in the framework of the standard working group of IAEA (the so-called reference file). However, some discrepancies, of up to 4%, are still present in some specific energy regions. The newdataset here presented, characterized by a unique combination of high resolution and accuracy, low background and wide energy range, can help to improve the evaluations from the Resolved Resonance Region up to 10 keV, also reducing the uncertainties that affect this region., CERN (European Organization for Nuclear Research)

Aeromonas species are found in the aquatic environment, drinking water, bottled mineral water, and different types of foods, such as meat, fish, seafood, or vegetables. Some of these species are primary or opportunistic pathogens for invertebrates and vertebrates, including humans. Among the pathogenic factors associated with these species, there are the lipopolysaccharides (LPSs). LPSs are the major components of the external leaflet of Gram-negative bacterial outer membrane. LPS is a glycoconjugate, generally composed of three portions: lipid A, core oligosaccharide, and O-specific polysaccharide or O-antigen. The latter, which may be present (smooth LPS) or not (rough LPS), is the most exposed part of the LPS and is involved in the pathogenicity by protecting infecting bacteria from serum complement killing and phagocytosis. The O-antigen is a polymer of repeating oligosaccharide units with high structural variability, particularly the terminal sugar, that confers the immunological specificity to the O-antigen. In this study, we established the structure of the O-chain repeating unit of the LPS from Aeromonas bivalvium strain 868 ET (=CECT 7113T = LMG 23376T), a mesophilic bacterium isolated from cockles (Cardium sp.) and obtained from a retail market in Barcelona (Spain), whose biosynthesis core LPS cluster does not contain the waaE gene as most of Aeromonas species. After mild acid hydrolysis, the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by chemical analysis and NMR spectroscopy. The polymer consists of a heptasaccharide repeating unit containing D-GalNAc, L-Rha, D-GlcNAc, and D-FucNAc residues.

Molecular recognition is a crucial issue in studies on interactions between biomolecules, or potential pharmacological agents and biomolecules. Isatin-hydrazones are interesting compounds since the presence of an imine bond combined with a set of heteroatoms lead to the formation of numerous isomers whose distribution can be modulated by solvent polarity, concentration, and pH range, among other factors. Herein we report NMR spectroscopic studies, complemented by UV/Vis and IR, to characterize geometrical isomers and tautomers of two isatin-derived hydrazones, isahim and isahpy. By using different spectroscopic methods, it was possible to detect and identify among diverse possible configurations of isahim molecule the preferential species and verify its stability in solution at different temperatures. NMR spectra indicated the co-existence of diverse isomer and/or tautomer forms in solution, corroborated by UV/VIS spectra in different solvents, at varied concentrations and pHs. Further, IR spectra allowed to better identify and characterize the proposed structures. DFT simulations supported the experimental results, attesting that the most stable isomers in solution are the keto in corresponding Z-forms for both hydrazone compounds. These studies provided suitable spectroscopic tools to understand and elucidate molecular interactions of such hydrazones with amyloid peptides and with essential metal ions implicated in Alzheimer disease, in subsequent studies.

In this paper, a complete 1 H and 13 C NMR data assignment of ent-polyalthic acid, a biologically active labdane-type diterpene, is presented. The assignments were carried on the basis of spectroscopic data from 1 H NMR, 13 C{1 H} NMR, gCOSY, gHMQC, and gHMBC experiments. Furthermore, a software-assisted methodology, using FOMSC3_rm_NB and NMR_MultSim programs, supported the detailed and unequivocal assignment of 1 H and 13 C signals, allowing all hydrogen coupling constants to be determined and thus clarifying all hydrogen signal multiplicities.

Orientadores: Nelci Fenalti Hoerhr, Roberto Rittner Neto Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas Resumo: Esse estudo analisou as propriedades toxicológicas de novos compostos organofosforados. Foram realizados experimentos para avaliar a atividade anticolinesterásica desses organofosforados, in vitro, no sangue total através do método de Ellman modificado. Para determinar a sua citotoxicidade foram utilizadas células PC 12, com as quais avaliamos a viabilidade celular após contato com os organofosforados e determinamos a IC 50, encontrando valores muito diferentes para os diversos organofosforados estudados. Estudos de toxicidade aguda in vivo foram realizados com camundongos, através da metodologia recomendada pela OECD nos quais determinamos a DL50 para três dos organofosforados estudados, sendo que um apresentou toxidade moderada. Foram analisados os efeitos dos solventes nas constantes de acoplamento JHH, JHH, JNC e 2Jnc em espectros de RMN de LH e 13C do cloreto de acetilcolina. Os valores das constantes de acoplamento em solventes de diferentes constantes dielétricas (s) não sofreram variações, indicando uma ausência de efeitos de solvente no equilíbrio conformacional do cloreto de acetilcolina (ACh). As constantes de acoplamento mostram que o sistema OCH2CH2N+ tem uma conformação gaúche (sinclinal). O Jnh e Jkc são observados na maioria dos solventes, mas não em solventes clorados e não são dependentes da viscosidade do solvente, esse comportamento foi explicado usando dados de medidas de Ti. Os valores dos coeficientes de difusão de RMN mostraram que a ACh tem uma grande tendência de se agregar quando dissolvida em solventes clorados, fato que pode explicar as diferenças observadas em valores de T1 para o 14N Abstract: This study analyzed the properties of the news organophosphorus. Experiments had been carried through to evaluate the inhibition of acetylcholinesterase of these organophosphorus, in vitro, through the modified EUman's method. In order to determine its cytotoxicity cells PC 12 had been used, with which we evaluate the cellular viability after contact with the organophosphorus and determined the IC50, different values were found for the diverse organophosphorus. Studies of acute toxicity had been carried through with mice, following the methodology recommended by the OECD in which determine the DL50 for three of the organophosphorus studied, being that one presented moderate toxicity. Coupling constants values ( Jhh and Jnc) obtained from the 'H and 13 C NMR spectra of acetylcholine chloride (ACh) in several solvents with a wide range of dielectric constants (e) are remarkably invariant, indicating an absence of solvent effects in the conformational equilibrium of this compound. Those values show that the OCH2CH2N+ system occurs in a synclinal conformation. The Jnh and Jnc are observable in most solvents, but not in chlorine-containing solvents and are not dependent on solvent viscosity. This behavior was explained using data from Ti measurements. The measurement of NMR diffusion coefficients show that ACh has a greater tendency to aggregate when dissolved in chlorinated solvents, a fact that could explain the observed differences in 14N T1 Mestrado Patologia Clínica Mestre em Ciências Médicas

Orientador: Gabriela Castellano Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: O conectoma cerebral refere-se ao mapeamento dos circuitos neurais com os objetivos de 1) identificar regiões que dão suporte às atividades mentais e comportamentais, e 2) detectar alterações nesses circuitos que levam a distúrbios de ordem psiquiátrica e neurológica. Na prática, os estudos de conectoma cerebral consistem na integração de técnicas multimodais de imageamento como ressonância magnética (RM), eletroencefalograma (EEG) e magnetoencefalograma (MEG) com o intuito de estimar os tipos e os níveis de conexão entre regiões cerebrais remotas. Essa "conectividade" entre regiões cerebrais é geralmente classificada em três tipos: anatômica, funcional e efetiva. No presente trabalho, as técnicas de conectividade, usando dados de MR, foram aplicadas na comparação de grupos saudáveis e patológicos. Pela técnica de conectividade anatômica observou-se anomalias na substância branca de pacientes com mutação no gene SPG11. Essa anomalias foram detectadas através da redução da anisotropia fracional (FA) e aumento da difusividade média (MD), difusividade radial (RD) e difusividade axial (AD) em regiões subcorticais dos lobos temporal e frontal, bem como no giro do cíngulo, cuneus striatum, corpo caloso e tronco cerebral. Tais achados indicam que o dano neuronal é mais difuso do que indicava a literatura. Um segundo estudo de conectividade anatômica demonstrou que esses índices de difusividade não foram robustos para diferenciar idosos com e sem diagnóstico de depressão indicando a necessidade de avanços na formulação de novos índices com maior sensibilidade. A técnica de conectividade funcional foi empregada em três estudos. No primeiro, observou-se que pacientes com epilepsia de lobo temporal medial unilateral apresentam redução da conectividade funcional durante a execução de tarefas de memória verbal e visual. Essa redução foi predominantemente ipslateral à lesão e associada ao material-específico utilizado no teste de memória. No segundo estudo, verificou-se uma redução dos padrões de conectividade funcional hipotalâmica em sujeitos obesos e a sua parcial elevação após a cirurgia bariátrica concomitantemente à redução de indicadores bioquímicos de inflamação. No terceiro estudo, observou-se que pacientes com doença de Alzheimer apresentaram elevação dos níveis de conectividade funcional na rede saliente (Salience Network) e redução na rede de modo padrão (Default-mode network). Adicionalmente, verificou-se nos pacientes a correlação positiva da síndrome hiperativa com os níveis de conectividade funcional no cíngulo anterior e em áreas da ínsula direita. O conjunto desses resultados ilustra um possível significado clínico para futuro diagnóstico e tratamento da doença de Alzheimer. Pela técnica de conectividade efetiva observou-se que em função do envelhecimento sadio há uma mudança dos parâmetros de conectividade durante a codificação de palavras com conteúdo emocional. A influência do hipocampo sobre a amígdala ipslateral é reduzida nos sujeitos mais velhos enquanto a influência da amígdala direita sobre o hipocampo direito é elevada. Tais achados reforçam a tese da ininterrupta plasticidade etária e da dinâmica cerebral normal. Essa mesma técnica foi também empregada para demonstrar os diferentes padrões de influência entre os lobos frontal e temporal de pacientes com ELTM esquerda e sujeitos controle. Encontrou-se alteração nos padrões de conectividade efetiva dos pacientes, indicando que estes podem ser potenciais biomarcadores para a epilepsia Abstract: Connectome refers to the neural circuitry mapping aiming to identify brain regions that support mental and behavioral functions as well as to detect circuit changes that are linked to psychiatric or neurologic disorders. In practice, connectome studies link several neuroimaging approaches such as MRI, EEG and MEG by means of the estimation of connections among remote brain regions. This "connectivity" among brain regions is usually classified as anatomic, functional or effective. In this work, the technique of connectivity, using MR data, was applied to compare healthy and pathological groups. By means of the anatomical connectivity abnormalities in the white matter of patients with SPG11 mutation were observed. These abnormalities were expressed as the reduction of the levels of fractional anisotropy (FA) and the increase in mean (MD) and radial diffusivities (RD) in sub-cortical regions of temporal and frontal lobe as well as in cingulated gyrus, cuneus, striatum, corpus callosum and brainstem. These findings suggest that neuronal damage/dysfunction is more widespread than previously recognized in this condition. Another anatomical connectivity study showed that such indices of diffusivity were not robust to statistically differentiate between old subjects with and without depression. This lacking on finding differences between both groups indicates that new indices of diffusivity have to emerge in order to provide complementary information about brain subtle microstructures. Functional connectivity was applied to three studies. In the first study, it was observed that patients with unilateral medial temporal lobe epilepsy presented lower levels of functional connectivity during visual or verbal memory tasks. Such reduction was ipsilateral to the side of the lesion and associated to the specific-material used in the memory task. In the second work, the levels of functional connectivity were reduced in hypothalamic regions of obese patients but a partial reversibility of hypothalamic dysfunction was observed after bariatric surgery. In the third, patients with Alzheimer disease presented higher values of functional connectivity in the salience network and a reduction of connectivity values in the default-mode network. Also in these patients, significant correlations between the levels of hyperactivity syndrome and the salience network were observed in the anterior cingulate cortex and right insula areas. These results indicate the potential clinical significance of resting state alterations in future diagnosis and therapy of Alzheimer disease. The effective connectivity approaches demonstrated that old and young subjects have significant differences when encoding words with emotional contents. The influence of the hippocampus on the ipsilateral amygdale was lower for older subjects whereas the influence of the right amygdale on the right hippocampus was increased for these subjects. These findings suggest that brain plasticity also happens as function of age. The same approach was used to estimate the influence from frontal to temporal lobes in patients with left MTLE compared to healthy subjects. The patterns of effective connectivity were changed in patients and may be potentially considered as biomarkers for epilepsy Doutorado Neurociências Doutor em Ciências

Orientador: Ljubica Tasic Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química Resumo: Esta tese de doutorado apresenta resultados pioneiros de estudos lipidômicos de alguns peixes amazônicos. Foram estudadas nove espécies de peixes, as quais podem ser classificadas em cinco hábitos alimentares: onívoros, detritívoros, piscívoros, planctívoros e carnívoros. Os lipídios foram extraídos do fígado e do músculo dorsal de peixes selvagens, coletados durante dois anos consecutivos nos períodos da cheia e da seca do Amazonas. Duas abordagens de análise lipidômica foram usadas. Na primeira, as análises quali- e quantitativas, aplicando a técnica de Espectroscopia de Ressonância Magnética Nuclear foram executadas. Nesta investigação, os principais lipídios foram identificados, o que possibilitou, através de uma análise quimiométrica, a determinação de potenciais biomarcadores por fenótipo. A relação entre ácidos graxos 'omega'-6 e 'omega'-3, com baixas quantidades de colesterol, foi observada como característica comum entre os peixes analisados, com a destacada presença de cardiolipinas em apenas amostras de fígados, sendo que a exceção foi o músculo dos piscívoros. Na segunda abordagem, empregando método de Espectrometria de Massas, a composição fosfolipídica dos peixes amazônicos foi avaliada, sendo encontrados 63 fosfolipídios diferentes. Uma maior quantidade e diversidade estrutural foi observada para a classe de glicerofosfocolinas, sendo que estudos de quimiometria mostraram que a sua composição não é influenciada pelos períodos sazonais, seca ou cheia do rio, fator associado a quantidade de alimentos disponível e nem pelo hábito alimentar. Contudo, uma maior diferença foi observada na composição de glicerofosfocolina quando seus tecidos de extração, fígado ou músculo, são comparados. E ainda, como resultado dessa investigação, alguns fosfolipídios foram apontados como interessantes candidatos contra doenças do sistema nervoso central, os quais estão envolvidos principalmente nos metabolismos de ácido linoléico e de eicosanóides Abstract: This thesis shows the pioneer research in lipidomics analysis of some Amazonian fishes. Nine species of fish were studied, which can be classified into five types according to their eating habits: omnivores, detritivores, piscivoros, plankivores and carnivores. Lipids were extracted from the livers and the dorsal muscles of wild fishes collected during two consecutive years, flood and drought periods of Amazonas. Two approaches to lipidomics were used. In the first, the qualitative and quantitative analyzes, applying the Nuclear Magnetic Resonance Spectroscopy technique were performed. In this investigation, the main lipids were identified, which enabled through a chemometric analysis, to determine potential biomarkers for fish phenotypes. The ratio between 'omega'-6 and 'omega'-3 fatty acids, with low amounts of cholesterol, was observed as a common characteristic among analyzed fish, with the outstanding presence of cardiolipins in only some of these species, principally, their liver tissues, with only one muscle tissue being rich in cardiolipins. In the second approach, using the Mass Spectrometry methods, the phospholipid composition of the Amazonian fish was evaluated and 63 different phospholipids were found. A greater amount and structural diversity was observed for the glycerophosphocholines class. And chemometrics studies showed that lipids composition is not influenced by the seasonal, drought or flood seasons of the river, which is a factor associated with the amount of food available, and not even by dietary habits. However, a greater difference was observed in the composition of glycerophosphocholine when tissues, liver or muscle, from where those were extracted were compared. Also, as a result of this investigation, some phospholipids have been identified as interesting candidates against diseases of the central nervous system, which are mainly involved in the metabolism of linoleic acid and eicosanoids Doutorado Química Inorgânica Doutora em Ciências FAPESP 2013/14707-9 CAPES

Orientador: Ljubica Tasic Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química Resumo: Esta tese de doutorado apresenta os resultados obtidos no estudo metabolômico de ovelhas saudáveis e infectadas pela Corynebacterium pseudotuberculosis, agente etiológico da Linfadenite Caseosa, doença que acomete principalmente ruminantes, levando à morte desses animais e prejuízos econômicos no setor agropecuário. Nosso estudo visou a identificação de biomarcadores da Linfadenite Caseosa usando a técnica de espectroscopia de Ressonância Magnética Nuclear (RMN) em conjunto com a quimiometria aplicados à sessenta amostras de soro de sangue desses animais (27 saudáveis + 33 infectados), de modo inédito. Foram identificados cerca de vinte metabólitos-chave sendo nove específicos no perfil controle saudável (animais soronegativos) e cinco nos doentes (animais soropositivos), em que esses últimos oriundos do metabolismo da bactéria e produzidos em processos de fermentação e degradação da parede bacteriana. A segunda parte da nossa pesquisa apresenta o estudo do comportamento da Corynebacterium pseudotuberculosis frente a ação da solução de nanopartículas de prata biogênicas obtidas a partir da síntese extracelular do fungo Fusarium oxysporum, para isso foram feitos ensaios para determinação da Concentração Inibitória Mínima (CIM) e análise por Microscopia Eletrônica de Varredura (MEV) do patógeno com e sem a presença das nanopartículas de prata. Finalmente, foram desenvolvidas junto com os integrantes do nosso grupo, pomadas com as nanopartículas de prata biogênicas, que têm sido aplicadas nos ferimentos dos animais doentes e tem apresentado promisores resultados no processo de cicatrização, em um curto período de tempo. Dessa forma, a nossa pesquisa abre perspectivas para um novo estudo bioquímico da atuação desse creme e que indica a técnica de RMN propícia para o desenvolvimento de um novo método de diagnóstico da doença, uma vez que o preparo de amostra é bem pouco invasivo (apenas 250 mL do soro de sangue) e dispendioso em relação ao método atual de Polymerase Chain Reaction (PCR) Abstract: This PhD thesis presents the results obtained from the metabolomics study of healthy and infected sheep by Corynebacterium pseudotuberculosis, the etiologic agent of Caseous Lymphadenitis, a disease that mainly affects ruminants, leading to the death of these animals and great economic losses in the agribusiness. The aim of our research was to identify biomarkers for Caseous Lymphadenitis using the Nuclear Magnetic Resonance (NMR) spectroscopy technique and chemometrics applied on sixty blood serum samples from the 27 healthy and 33 infected animals, comprising an inedited approach. Around twenty key metabolites were identified, nine of which were specific for the healthy control profile (seronegative animals) and five in the seropositive animals, the last being derived from the metabolism of the bacteria and produced in fermentation and degradation processes. The second part of our research was the study of the behavior of Corynebacterium pseudotuberculosis when put in contact with the solution of biogenic silver nanoparticles obtained in extracellular synthesis from the fungus Fusarium oxysporum. Determination of the Minimal Inhibitory Concentration (MIC) and Scanning Electron Microscopy (SEM) analyses of the pathogen in the absence and in the presence of silver nanoparticles were conducted. Finally, we developed, a cream based on biogenic silver nanoparticles, which has been applied in the wounds of the LC sick animals and has shown very promising results in the healing processes in a short period. Thus, our research opens perspectives for a new biochemical study of the performance of this cream and indicates the NMR ¿based metabolomics as suitable for the development of a new method of diagnosis of the disease, since sample preparation is not very invasive (only 250 ?L of serum) and less costly than the current method ¿ the Polymerase Chain Reaction (PCR) Doutorado Química Orgânica Doutor em Ciências CNPQ 147952/2013-1

Functional tea beverages have emerged as a novel approach to achieving health benefits associated with tea. The use of metabolomics may improve the evaluation of their consumption and their effects. The current study aimed at exploring the urinary signature of exposure to a functional high-catechin tea (HCT) using untargeted NMR-based metabolomics. Ten volunteers participated in a crossover intervention study. Individuals consumed an HCT or a control beverage over a period of 28 days. Multilevel partial least-squares discriminant analysis (ML-PLS-DA) was used for paired comparisons. A further crossover model was performed to assess the significant changes. The consumption of the HCT resulted in the excretion of theanine, epicatechin, pyrogallol sulfate, and higher levels of 3-methyl-2-oxovalerate and succinate, as well as unknown compounds. In conclusion, the present work established novel urinary signatures of a functional drink. Such signatures may be potential biomarkers and/or reflect certain benefits of functional tea beverages.

Orientador: Leticia Rittner Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação Resumo: A espectroscopia por ressonância magnética (MRS), apesar de ser uma técnica recente, vem sendo bastante utilizada para estudar alterações metabólicas em tumores cerebrais e outras doenças do cérebro em geral. Porém ainda não existem métodos que explorem bem as informações contidas na região da imagem de ressonância magnética de onde o conjunto de espectros foi adquirido. Conhecer essas informações é importante, pois permite que a quantificação dos metabólitos seja mais precisa. O presente trabalho tem como objetivo apresentar um método capaz de analisar a região da imagem de ressonância magnética de onde foi coletado o conjunto de espectros, identificando os tipos de tecidos: substância branca (WM - White matter), substância cinzenta (GM - Gray matter) e fluido cerebroespinal (CSF - Cerebrospinal fluid) e/ou estrutura cerebral (hipocampo, corpo caloso, etc.), nela contidos e, posteriormente, possibilitar a seleção de um subconjunto de espectros de interesse. Essa abordagem torna possível analisar melhor os dados adquiridos através da técnica de MRSI, agrupando espectros provenientes de regiões semelhantes, como tecidos (GM, WM e CSF) ou estruturas (estruturas cerebrais, tumores ou lesões). A partir deste agrupamento é possível criar subconjuntos de espectros de interesse e realizar a quantificação dos metabólitos em cada um dos espectros isoladamente ou todos os espectros do subconjunto Abstract: Magnetic Resonance Spectroscopy (MRS) although a recent technique, has been widely used to study metabolic changes in brain tumors and other diseases of the brain in general. Currently there are no good methods that exploit the information contained in the region of the magnetic resonance image, from where the set of spectra was acquired. This information is important to allow a more accurate quantification of metabolites. This study aims to present a method to analyze the region of the magnetic resonance image from where the set of spectra was collected, identifying the types of tissue (white matter, gray matter and cerebrospinal fluid) and/or brain structure (hippocampus, corpus callosum, etc.) contained, and consequently allowing the selection of a set of the spectra of interest. This approach makes it possible to better analyze the data acquired through the MRSI, grouping spectra from similar regions, such as tissues (GM, WM and CSF) or structures (brain structures, tumors or lesions). From this grouping it is possible to create subsets of spectra of interest and execute the quantification of the metabolites in each spectrum alone or in all the spectra of the subset Mestrado Engenharia de Computação Mestre em Engenharia Elétrica FAPESP

Orientador: Roberto Rittner Neto Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química Resumo: O entendimento das preferências conformacionais de aminoácidos e seus derivados ainda é um desafio, uma vez que diversos efeitos podem afetar as preferências conformacionais destes compostos. No presente trabalho é proposta a investigação de derivados esterificados e N-acetilados dos aminoácidos metionina e ácido glutâmico utilizando uma combinação de RMN de ¹H e cálculos teóricos de estrutura eletrônica como uma alternativa para o estudo de aminoácidos em meio condensado, uma vez que derivados de aminoácidos esterificados e N-acetilados são solúveis na maioria dos solventes orgânicos e representam uma boa aproximação do que acontece com um resíduo de aminoácido em uma cadeia polipeptídica. As geometrias e energias dos confôrmeros mais estáveis, tanto em fase isolada, quanto em solução (modelo de solvatação implícito IEF-PCM) foram obtidas em nível de teoria 'omega'B97X-D/aug-cc-pVTZ. O equilíbrio conformacional dos compostos em solução também foi avaliado através de dados de RMN utilizando valores de constantes de acoplamento escalar spin-spin ³JHH experimentais e teóricas em diferentes solventes orgânicos. Uma comparação entre os valores de ³JHH experimentais e calculados indicam que o equilíbrio conformacional do derivado esterificado da metionina (MetOEt) não se altera com a variação do solvente. Por outro lado, os confôrmeros do derivado N-acetilado da metionina (AcMetOEt) e dos derivados esterificado e N-acetilado do ácido glutâmico (GluOMe e AcGluOMe) apresentam variações nas populações quando a polaridade do meio é aumentada de CHCl3 para DMSO. De acordo com as análises de Orbitais Naturais de Ligação (NBO), da Teoria Quântica de Átomos em Moléculas (QTAIM) e de Interações Não Covalentes (NCI), verificou-se que as preferências conformacionais dos compostos estudados não são ditadas por uma interação específica, mas sim, por uma combinação entre interações estéricas e hiperconjugativas Abstract: The understanding of the conformational behavior of the amino acids and their derivatives is a challenge task, since several effects can affect their conformational preferences. In the present work, the conformational analysis of esterified and N-acetylated derivatives of methionine and glutamic acid using a combination of ¹H NMR spectroscopy and electronic structure calculations is reported as an alternative for the study of amino acids in solution. The studied derivatives are soluble in the most of organic solvents and thus, their study can represent a reasonable approximation to the behavior of an amino acid residue into a polypeptide chain. The geometries and energies of the most stable conformers in isolated phase and taking into account the implicit solvent effects, according to the integral equation formalism polarizable continuum model (IEF-PCM) were obtained at the 'omega'B97X-D/aug-cc-pVTZ level of theory. The conformational equilibrium of the compounds in solution were also determined from experimental and theoretical NMR data using ³JHH spin-spin coupling constants in different organic solvents. A comparison between the experimental and theoretical coupling constants indicate that the conformational stability of the esterified derivative of methionine (MetOEt) is not very sensitive to solvent polarity, whereas the conformers of the N-acetylated derivate of methionine as well as the esterified and N-acetylated derivates of glutamic acid (GluOMe and AcGluOMe) had their populations changed when the polarity of the medium was varied from CHCl3 to DMSO. According to the Natural Bond Orbital (NBO), Quantum Theory of Atoms in Molecules (QTAIM) and Noncovalent Interactions (NCI) analyses, the conformational preferences for the studied compounds are not dictated by an specific interaction, but due to a combination of hyperconjugative and steric interactions Mestrado Química Orgânica Mestre em Química FAPESP 2016/12005-5

Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor activities in multiple cancers such as glioblastoma, melanoma and others. Although most of the authors suggest that their mechanism of action comes from the activation of p53 and inactivation of NF-kB by targeting FACT, there is evidence supporting the involvement of DNA binding in their antitumor activity. In this work, the DNA binding properties of curaxin CBL0137 with model quadruplex DNA oligomers were studied by 1H NMR, CD, fluorescence and molecular modeling. We provided molecular details of the interaction of curaxin with two G-quadruplex structures, the single repeat of human telomere d(TTAGGGT)4 and the c-myc promoter Pu22 sequence. We also performed 1H and 31P NMR experiments were also performed in order to investigate the interaction with duplex DNA models. Our data support the hypothesis that the interaction of curaxin with G-quadruplex may provide a novel insight into the DNA-binding properties of CBL0137, and it will be helpful for the design of novel selective DNA-targeting curaxin analogues., This research was funded by PIANO DI SOSTEGNO ALLA RICERCA 2020—Linea 2 azione B (DEFENS) and by the Spanish Ministerio de Ciencia e Innnvacion (PID2019-107158GB-I00) and recognition from the Autonomous Catalan government (2017SGR114).

LiCH3 and LiCH2CH3 react with the complex [Mo2(H)2(μ-AdDipp2)2(thf)2] (1⋅thf) with coordination of two molecules of LiCH2R (R=H, CH3) and formation of complexes [Mo2{μ-HLi(thf)CH2R}2(AdDipp2)2], 5⋅LiCH3 and 5⋅LiCH2CH3, respectively (AdDipp2=HC(NDipp)2; Dipp=2,6-iPr2C6H3; thf=C4H8O). Due to steric hindrance, only one molecule of LiC6H5 adds to 1⋅thf generating the complex [Mo2(H){μ-HLi(thf)C6H5}(μ-AdDipp2)2], (4⋅LiC6H5). Computational studies disclose the existence of five-center six-electron bonding within the H−Mo≣Mo−C−Li metallacycles, with a mostly covalent H−Mo≣Mo−C group and predominantly ionic Li−C and Li−H interactions. However, the latter bonds exhibit non-negligible covalency, as indicated by X-ray, computational data and the large one-bond 6,7Li,1H and 6,7Li,13C NMR coupling constants found for the three-atom H−Li−C chains. By contrast, the phenyl group in 4⋅LiC6H5 coordinates in an η2 fashion to the lithium atom through the ipso and one of the ortho carbon atoms.

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2021, Tutor: Xavier Companyó Montaner, Asymmetric catalysis has been one of the main topics in organic chemistry in the last 50 years. Traditionally, asymmetric allylic alkylation reactions (AAA) were based on transitionmetal-catalysed reactions. Recently, the organocatalytic AAA has been developed where small organic molecules, such as chiral tertiary amines and phosphines, are used as catalysts instead of transition metals. The objective of the TFG has been the development of a new asymmetric allylic alkylation reaction (AAA) using Morita-Baylis-Hillman (MBH) fluorides and nucleophiles activated by the fluoride anion (difluoroalkenes). The leaving group of the MBH adduct activates the nucleophile, and it is incorporated in the final product as a trifluoromethyl group, this is the distinguishing aspect of the reaction. The products are interesting because of its trifluoromethyl group, a singular group in organic chemistry which is commonly used in pharmaceutical industry. The reactivity has been tested using chiral tertiary amines as catalyst, such as β-isocupreidine, a compound derived from quinine, to synthesise the corresponding enantioenriched products. The characterisation of the products has been performed by spectroscopic and chromatographic methods such as NMR and chiral HPLC, respectively

Fundação de Amparo à Pesquisa do Estado de Minas Gerais As lactonas sesquiterpênicas pertencem a um grupo de metabolitos secundários encontrados em plantas da família Asteraceae, e apresentam amplo espectro de atividades biológicas. Merecem destaque também os endoperóxidos, que são conhecidos por suas atividades citotóxicas e antimaláricas. A elucidação estrutural é um aspecto relevante, mas não é incomum de se encontrar na literatura atribuições estruturais incompletas ou que foram feitas de maneira incorreta. Cálculos baseados na mecânica quântica são frequentemente utilizados para computar deslocamentos químicos de RMN de 1H e de 13C, e depois utiliza-los em comparação com os dados experimentais a fim de se estabelecer a estrutura correta entre diversos candidatos. Visando a síntese de compostos bioativos, previu-se a síntese de um novo endoperóxido a partir da lactona sesqueterpênica α- santonina (1a). A transformação fotoquímica da α-santonina (1a) resulta na mazdassantonina (2a), seguido por foto-oxidação catalisado por Rosa de bengala da origem à um novo endoperóxido, com rendimento global de 23% a partir da α-santonina (1a). O endoperóxido sintetizado apresenta cinco centros estereogênicos e é um análogo do agente antimalárico artemisinina XXVIII. A estereoquímica dos centros estereogênicos do endoperóxido foram atribuídas através da análise dos espectros de RMN e por cálculos teóricos. Ambas abordagens estiveram de acordo e a estrutura do endoperóxido foi estabelecida como (3S,3aS,5aS,8R,9bS)-3,6,6-trimetil-3,3a,4,5,8,9b-hexahidro- 2H-5a,8-epidioxinafto[1,2-b]furan-2,7(6H)-dione (endo1). As 2H-cromen-2-onas, conhecidas como cumarinas, e mais especificamente as furanocumarinas, são metabolitos secundários encontrados em plantas da família Rutaceae, e apresentam diversas propriedades farmacológicas. Se inspirando nesta classe de compostos e nas lactonas sesquiterpênicas, objetivou se a síntese de dois análogos, um bromado e outro clorado, a partir do ácido fotossantônico (3a). O ácido foi sintetizado a partir da transformação fotoquímica da α-santonina (1a) em meio prótico, resultando no ácido fotossantônico (3a) com rendimento de 45%, e que por meio de uma reação de halolactonização utilizando Cl 2 ou Br 2 , resultou nos produtos bromados e clorados com rendimento de 95 e 86% respectivamente. Os produtos obtidos possuem 4 centros estereogênicos, e a configuração destes foram confirmados em conjunção com os dados de RMN, de cálculos teóricos, e por meio da análise de erro médio absoluto (EMA) e de DP4. Todas as abordagens estão de acordo e a estrutura dos produtos bromado e clorado foram definidas como (3S,3aS,5aR)-5a-(2- bromopropan-2-il)-3-metil-3,3a,5,5a,8,9b-hexahidro-4H-furo[2,3-f]cromeno-2,7-diona (BR2) e (3S,3aS,5aR)-5a-(2-cloropropan-2-il)-3-metil-3,3a,5,5a,8,9b-hexahidro-4H-furo[2,3-f]cromeno- 2,7-diona (CL2). Por último, cálculos teóricos indicaram os produtos BR2 e CL2 como os produtos termodinâmicos e cinéticos da reação. PALAVRAS-CHAVE: Lactonas sesquiterpênicas. Endoperóxido. Cromenodiona. Fotoquímica. RMN. Cálculo teórico. Sesquiterpene lactones are secondary metabolites found in plants of Asteraceae family and have a wide range of biological activities. Also worth mentioning are endoperoxides, which are known for their antimalarial activity. Structural elucidation is a relevant aspect; however, it is not uncommon to find mistakenly or incomplete structural assignments in the literature. Calculations based in quantum mechanics are frequently used to compute 1H and 13C NMR chemical shifts, and after comparing with the experimental data, to establish the correct structure from diverse candidates. Aiming the synthesis of bioactive compounds, we have envisaged the synthesis of a novel endoperoxide from the natural sesquiterpene lactone  -santonin (2). Photochemical transformation of α-santonin (2) to mazdassantonin (4) followed by photo-oxidation catalyzed by rose Bengal afforded a novel endoperoxide (endo1). The endoperoxide synthesized presents five stereogenic centers and is analogous to the antimalarial agent artemisinin (1). The relative stereochemistries of the stereogenic centers of the endoperoxide were established by NMR analyses and confirmed by theoretical calculations. All approaches were in complete agreement and the structure of mazdasantonin endoperoxide was established as (3S,3aS,5aS,8R,9bS)-3,6,6-trimethyl- 3,3a,4,5,8,9b-hexahydro-2H-5a,8-epidioxynaphtho[1,2-b]furan-2,7(6H)-dione. The class of 2H- Chromene-2-ones known as coumarins and specifically furanocoumarins, are metabolites found in high plants of the Rutaceae family, and they are known to possess several pharmacological proprieties. Inspired by this class of natural compounds and sesquiterpene lactones, this work aimed to synthesize two halogenated analogs, starting with photossantonic acid (3a). The acid was synthesized by a photochemical transformation of α-santonin (1a) in protic media, resulting in photossantonic acid (3a) with a 45% yield, and through halolactonization reaction using Cl 2 and Br 2 , afforded a brominated a chlorinated product in 95 and 86% yield respectively. The products obtained have four stereogenic centers, and these were confirmed with the conjunction of NMR data, theoretical calculations and by use of MAE and DP4 analysis. All approaches were in agreement and the structure of the brominated and chlorinated products was defined as (3S,3aS,5aR)-5a-(2-bromopropan-2-yl)-3-methyl-3,3a,5.5a,8,9b-hexahydro-4H-furo[2,3-f] chromene-2,7-dione (BR2) and (3S,3aS,5aR)-5a-(2-chloropropan-2-yl)-3-methyl-3.3a,5.5a,8.9b- hexahydro-4H-furo[2,3-f]chromene-2,7-dione (CL2). Finally, theoretical calculations indicated the products BR2 and Cl2 as the thermodynamic and kinetic products of the reaction. KEYWORDS: Sesquiterpene lactones. Endoperoxide. Chromendione. Photochemistry. NMR. Theoretical calculations.

Substâncias derivadas da biomassa têm chamado a atenção, uma vez que esses compostos pertencem ao grupo de solventes renováveis, cujas propriedades físico-químicas apresentam grande potencial para aplicações em diversas áreas da ciência e indústria, especialmente em biorrefinarias. Portanto, conhecer as propriedades termodinâmicas de sistemas contendo tais líquidos é de interesse prático, pois dados experimentais podem ser usados em modelagem a fim de aperfeiçoar processos ou ainda desenvolver e verificar novos modelos e teorias de soluções, as quais têm se mostrado bastante complexas. O presente projeto objetiva estudar propriedades termodinâmicas excesso, que quantificam o desvio da solução real em relação à ideal, e espectroscópicas de sistemas líquidos binários compostos por gama-valerolactona (GVL) + álcoois (etanol, 1-propanol, 1-butanol ou 1-pentanol). Para tanto, determinaram-se dados originais de densidade, velocidade do som e viscosidade dinâmica dessas soluções em toda faixa de composição e nas temperaturas T = (293,15 – 313,15) K, sob pressão atmosférica (92,3 kPa). Com estes dados experimentais, calcularam-se propriedades volumétricas como volume molar excesso (Vm E ), acústica tal como desvio da compressibilidade isentrópica (??S), viscosimétrica como desvio da viscosidade (??) e a energia de Gibbs de ativação de fluxo excesso (?G *E). Estas propriedades resultaram em valores negativos para todas as condições, exceto o Vm E , que foi totalmente negativo apenas para o sistema etanol-GVL. Para complementar a discussão termodinâmica, realizaram-se análises espectroscópicas por ressonância magnética nuclear (RMN-H 1 e RMN-C 13) e no infravermelho (ATR-FTIR), cujos espectros comprovaram a existência de ligação de hidrogênio fraca entre o GVL-álcool Substances derived from biomass have called attention, once these compounds belong to the group of renewable solvents, whose physicochemical properties present great potential for applications in several areas of science and industry, namely in biorefineries. Therefore, thermodynamic properties of systems containing such liquids is of practical interest, because experimental data can be used in modeling in order to improve processes or yet to develop and verify new models and solution theories, which have shown their complexity. The present project aims at studying excess thermodynamic properties, that quantify the real solution deviation in comparison to the ideal one, and spectroscopic analyses of binary liquid systems constituted of gamma-valerolactone (GVL) + alcohols (ethanol, 1-propanol, 1-butanol or 1- pentanol). For this purpose, original values of density, speed of sound and dynamic viscosity were determined for these solutions in the whole composition range and in temperatures T = (293.15 – 313.15) K under ambient pressure (92.3 kPa). From these experimental results, it was possible to calculate volumetric properties such as excess molar volume (Vm E ), acoustic property as deviation in isentropic compressibility (??S), viscometric as deviation in viscosity (??) and excess Gibbs energy of activation of viscous flow (?G *E). These properties resulted in negative values at all conditions, except for Vm E , which was totally negative only in ethanol-GVL system. To complete the thermodynamic discussion, both nuclear magnetic resonance (1H-NMR and 13C-NMR) and infrared (ATR-FTIR) spectroscopies have been performed, whose spectra proved the existence of weak hydrogen bondings between GVLalcohol

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2020, Tutora: Margarita Crespo Vicente, In this work, the synthesis of several platinum (II) cyclometallated compounds with bidentate ligands type [C,N] has been reported. In order to carry out this synthesis, compound K2[PtCl4] has been used to prepare the metal agent (1a) used in all cyclometallation reactions. One amine (2a) and three different imines (2b-2c), previously synthesized from amine 2a and the corresponding aldehyde, have been used as ligands. These ligands have been reacted with compound 1a in toluene or in methanol to inquire which of these solvents is more optimal for the cyclometallation, thus obtaining cyclometallated compounds (3a-3d). The expected isomer for this synthesis is the endo configuration but depending on the solvent and the imine used different isomers such as exo configuration or even cyclometallated compounds with metallacycles containing more than five members might be obtained. The latter were subsequently reacted with triphenylphosphane to form the corresponding derivatives (4a-4d) where the number of isomers has been reduced due to the stability given to the compound by triphenylphosphane. The nature of the synthesised compounds has been studied by proton nuclear magnetic resonance spectroscopy and for some compounds also by mass spectrometry

Most porous polymers are notoriously hard to characterize due to their amorphous and completely insoluble nature. On the other hand, they are an interesting class of materials for sorption, catalytic, and electrode applications, thus they warrant in-depth studies. In this contribution, we elaborate on the possibilities that dynamic nuclear polarization offers towards the investigation of the structure of porous polymers. We discuss the advantages and disadvantages of this technique in the investigation of model polymers.

Orientador: Gabriela Castellano Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin Resumo: Epilepsia do Lobo Temporal Mesial é o tipo de epilepsia mais frequente em adultos. Uma das causas associadas é a esclerose hipocampal, uma atrofia que consiste em perda de células nervosas do hipocampo. O tratamento inicia-se com a aplicação de drogas anti-epilépticas e pacientes que não possuem uma resposta adequada a esses medicamentos (farmacorresistentes) são candidatos aos procedimentos cirúrgicos. Aproximadamente um terço dos pacientes não possuem uma resposta adequada a medicamentos anti-epilépticos. O objetivo deste estudo é verificar se aprendizado de máquina, um ramo da inteligência artificial em que algoritmos encontram padrões por meio de dados fornecidos, pode ser aplicado na tentativa de prever pacientes portadores de epilepsia temporal mesial que possuem resistência às drogas anti-epilépticas, a partir de dados de espectroscopia de ressonância magnética. Um conjunto de dados de 115 pacientes com epilepsia temporal mesial (que posteriormente foi reduzido a 107 por critérios de exclusão) contendo indivíduos com e sem esclerose hipocampal foram obtidos de um projeto maior. Os metabólitos N-acetil-aspartato, colina, mio-inositol e o grupo formado por glutamato e glutamina foram quantificados dos espectros de ambos hipocampos e normalizados com relação a quantificação de creatina. Três abordagens de construção de vetores de atributos foram testadas: sobreposição da quantificação do lado com esclerose hipocampal, inclusão de duas variáveis para representar a presença ou não de esclerose no lado direto e esquerdo, e utilização da quantificação apenas do hipocampo com esclerose como vetor de atributos. Três algoritmos de aprendizado de máquina, Regressão Logística, Florestas Randômicas e Máquinas de Vetores de Suporte, foram explorados variando hiperparâmetros na busca pela combinação que resultasse em maior desempenho, medido pela área sob a curva (AUC) das respectivas curvas ROC (do inglês, receiver operating characteristic) e avaliado utilizando validação cruzada. Os melhores resultados obtidos foram utilizando somente a quantificação do hipocampo com esclerose como vetor de características, com os algoritmos Máquinas de Vetores de Suporte com o kernel RBF (5 folds, Gama=100 e C=0.01) e com o kernel sigmoidal (10 folds, Gama=0.01, Kappa=10, C=100) e Regressão Logística (10 folds, regularização L1 e C=1), que resultaram em AUC’s de 0.7 ± 0.1, e com os algoritmos Florestas Randômicas utilizando o critério de entropia (10 folds, 10 árvores e profundidade de 10 nodos) e utilizando o índice de Gini (10 folds, 20 árvores e máximo de 70 nodos) e Regressão Logística com regularização L2 (10 folds e C=0.1) obtendo uma AUC de 0.7 ± 0.2. Estes resultados indicam que esses algoritmos têm potencial para a previsão de farmacorresitência de pacientes portadores de epilepsia do lobo temporal mesial a partir concentração metabólica obtida por meio da espectroscopia de ressonância magnética dos hipocampos desses pacientes Abstract: Mesial Temporal Lobe Epilespy is the most frequently observed epilepsy in adults. One of the associated causes is hippocampal sclerosis, an atrophy that consists of loss of nerve cells in the hippocampus. Treatment begins with the application of anti-epileptic drugs, and patients who do not have an adequate response to this these drugs (pharmacoresistant) are candidates for surgical procedure. Approximately one third of patients do not have an adequate response to anti-epileptic drugs. The aim of this study is to verify whether machine learning, a branch of artificial intelligence in which algorithms find patterns through data, can be applied in an attempt to predict mesial temporal lobe epilepsy patients who are resistant to anti-epileptic drugs, using magnetic resonance spectroscopy data. A dataset of 115 mesial temporal lobe epilepsy patients (which was later reduced to 107 by exclusion criteria) containing individuals with and without hippocampal sclerosis was obtained from a larger project. The metabolites N-Acetyl-Aspartate, choline, myo-Inositol, and the glutamate and glutamine group were quantified from the spectra on both hippocampi and normalized in relation to the creatine quantification. Three approaches of feature vector construction were tested: superposition of the quantified side with hippocampus sclerosis, inclusion of two variables to represent the presence or not of hippocampal sclerosis in the right and left sides, and using only the quantification of the hippocampus with sclerosis as feature vector. Three machine learning algorithms, Logistic Regression, Random Forests and Support Vector Machines, were explored by varying hyperparameters in the search for the combination that would result in the best performance, measured by the area under the curve (AUC) of the respective ROC (receiver operating characteristic) curves and evaluated using crossvalidation. The best results were obtained using only the quantification of the side with hippocampal sclerosis, with the algorithms Support Vector Machines with the RBF kernel (5 folds, gamma=100 and C=0.01) and the sigmoidal kernel (10 folds, gamma=0.01, kappa=10, C=100) and Logistic Regression (10 folds, L1 Regularization and C=1), that resulted in an AUC of 0.7 ± 0.1, and with the algorithms Random Forests with the entropy criteria (10 folds, 10 trees and maximum depth of 10 nodes) and using the Gini index (10 folds, 20 trees and maximum depth of 70 nodes) and Logistic Regression with L2 regularization (10 folds and C=0.1) obtaining an AUC of 0.7 ± 0.2. These results indicate that these algorithms have the potential to predict the pharmacoresistance of patients with mesial temporal lobe epilepsy from the metabolic concentration obtained by magnetic resonance spectroscopy of the hippocampi of these patients Mestrado Física Aplicada Mestre em Física CAPES 1765883/2017

Palladium C–H bond activation in azobenzenes with R1 and R2 at para-positions of the phenyl rings (R1=N(Me)2, R2=H (L1); R1=N(Me)2, R2=Cl (L2); R1=N(Me)2, R2=I (L3); R1=–N(Me)2, R2=NO2 (L4); R1=H, R2=H (L5)) and their already monopalladated derivatives, using cis-[PdCl2(DMF)2] as precursor, has been studied in detail by in situ 1H NMR spectroscopy in N,N-dimethylformamide-d7 (DMF-d7) at room temperature; the same processes have been monitored in parallel via time-resolved UV-Vis spectroscopy in DMF at different temperatures and pressures. The final goal being to achieve, from a kinetico-mechanistic perspective, a complete insight of previously reported reactivity results. As expected, the results suggest the operation of an electrophilic substitution intimate mechanism for both the mono- and dipalladation reactions, occurring from the coordination compound and the monopalladated intermediates, respectively. The process involves a deprotonation of the C–H bond, which is assisted by the presence of a coordinated DMF molecule, that acts as a base. For the first time, NMR monitoring provides a direct evidence of all the intermediate stages, that is: i) coordination of the azo ligand to a PdII center, ii) formation of the monopalladated spe-cies, iii) coordination of the monopalladated species to another PdII unit, which finally result in the iv) formation of the dipalladated product. All of these species have been identified as intermediates in the dipalladation of azobenzenes, evidenced also by UV-Vis spectroscopy time-resolved monitoring; the data also confirms that the cyclopalladation of asymmetrically substituted azobenzenes occurs by two concurrent reaction paths. In order to identify the species observed by NMR and by UV-Vis spectroscopies, the final products, intermediates and the PdII precursor have been prepared and characterized by X-ray diffraction, IR and NMR spectroscopies. All attempts to isolate the intermediate monopalladated complexes attached to another PdII unit proved to be unsuccessful. DFT calculations have also been used in order to explain the isomerism ob-served for the isolated complexes, as well as to assign their NMR spectra.

Na3GaF6and Na3GaF6:Mn4+samples were obtained from NaNO3and Ga(NO3)3·9H2O in hydrofluoric acid using K2MnF6or NaMnO4as manganese sources. The structure of Na3GaF6was studied by single crystal X-ray diffraction at 90, 293, 440 and 500 K, confirming the monoclinic cryolite type structure, space groupP21/c. The gallium atoms show slightly distorted octahedral coordination by fluorine atoms, similar to the Na1 atoms. Coordination number 8 is observed for Na2. Both sodium sites are clearly distinguished by23Na MAS-NMR spectroscopy. Above 400 K the spectra reveal distinct chemical exchange effects, signifying sodium ion hopping between these two sites. At the same time static19F NMR spectra indicate pronounced motional narrowing effects in this temperature region. The nearly invariant69Ga MAS-NMR spectra suggest that any reorientational motion involving the GaF63−ions (if present) occurs with preservation of the center of mass of these octahedra.

A polycrystalline sample of the TiNiSi type low-temperature (LT) modification of ScRuSi was synthesized by arc-melting. Longer annealing in a sealed silica tube (6 weeks at 1270 K) followed by quenching led to the high-temperature (HT) phase. HT-ScRuSi adopts the ZrNiAl structure type: P 6 ¯ 2 m , a = 688.27(9), c = 336.72(5) pm, w R 2 = 0.0861, 260 F 2 values, 14 variables. The striking structural building units are regular, tricapped trigonal prisms Si1@Ru 3 Sc 6 and Si2@Ru 6 Sc 3 . Both polymorphs have been characterized by 29 Si and 45 Sc MAS-NMR spectroscopy. The local scandium environments in the two polymorphs are easily distinguished by their electric field gradient tensor values, in agreement with theoretically calculated values.

The new equiatomic silicides NbOsSi and TaOsSi as well as ZrOsSi, T IrSi ( T = Zr, Hf, Nb, Ta) and T PtSi ( T = Nb, Ta) were prepared from the elements by arc-melting. These silicides crystallize with the orthorhombic TiNiSi type structure, space group Pnma . Irregularly shaped crystals of ZrOsSi, NbOsSi, TaOsSi, ZrIrSi and HfIrSi were separated from the annealed samples and investigated by single-crystal X-ray diffraction ( a = 640.46(7), b = 404.07(5), c = 743.66(8) pm, wR 2 = 0.0285, 390 F 2 values, 20 variables for ZrOsSi; a = 629.78(6), b = 388.72(4), c = 727.48(7) pm, wR 2 = 0.0350, 397 F 2 values, 20 variables for NbOsSi, a = 626.80(6), b = 389.36(4), c = 726.22(7) pm, wR 2 = 0.0501, 385 F 2 values, 20 variables for TaOsSi, a = 653.48(8), b = 395.35(4), c = 739.19(8) pm, wR 2 = 0.0427, 413 F 2 values, 20 variables for ZrIrSi and a = 646.34(12), b = 393.57(7), c = 736.8(14) pm, wR 2 = 0.0582, 371 F 2 values, 20 variables for HfIrSi). The striking structural motifs in the new osmium compounds are three-dimensional [OsSi] networks (Os–Si: 240–251 pm) in which the osmium atoms have strongly distorted tetrahedral silicon coordination. High-pressure/high-temperature experiments (9.5 GPa/1520 K) on TaOsSi gave no hint for a structural phase transition. Temperature dependent measurements of the magnetic susceptibility and the electrical conductivity of NbOsSi and TaOsSi showed superconductivity below T C = 3.5 and 5.5 K, respectively. 29 Si solid state MAS NMR investigations of the prepared silicides approved the structural models and showed a correlation between the observed 29 Si resonance shifts and the electronegativity of the involved refractory metal.

The incarceration of o‐benzyne and 27 other guest molecules within hemicarcerand 1, as reported experimentally by Warmuth, and Cram and co‐workers, respectively, has been studied by density functional theory (DFT). 1H‐NMR chemical shifts, rotational mobility and conformational preference of the guests within the supramolecular cage were determined, which showed intriguing correlations of the chemical shifts with structural parameters of the host‐guest system. Furthermore, based on the computed chemical shifts reassignments of some NMR signals are proposed. This affects in particular the putative characterization of the volatile benzyne molecule inside a hemicarcerand, for which our CCSD(T) and KT2 results indicate that the experimentally observed signals are most likely not resulting from an isolated benzyne within the supramolecular host. Instead, we show that the guest reacted with an aromatic ring of the host, and this adduct is responsible for the experimentally observed signals The following organizations are thanked for financial support: MICINN (PhD-scholarship ACC CTQ2011-25086/BQU, projects CTQ2014-59212-P, CTQ2015-70851-ERC, CTQ2017-87392-P), CONACyT project 2014-383560, GenCat (PhD scholarship ARR 2015_FI_B_00165, 2014SGR1202 and XRQTC network) and European Fund for Regional Development (FEDER, UNGI10-4E-801)

Colorectal Carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. In order to reduce the morbidity and mortality of this disease, screening techniques for asymptomatic patients are extensively studied in the literature. Biopsy by videocolonoscopy is widely used, but presents a considerable rate of morbidity inherent in invasive diagnostic procedures. In view of the above, metabolomics have emerged as a viable alternative and with acceptable sensitivity and specificity for CRC diagnosis. The metabolic study of faecal material has been shown to be more effective because of its proximity to the mucosa of the large intestine and because it is a direct product of interactions between dietary compounds and intestinal microbiota. In an attempt to distinguish a metabolic pattern of healthy patients in relation to patients with CRC, fecal analysis of these distinct groups was performed by magnetic resonance spectroscopy (MRE). Objectives: to differentiate a typical metabolic profile of CRC patients versus the control group (CG) and to compare these results with a systematic review of theliterature (SRL). Method: 11 patients from GC and 5 patients with CRC were selected at the HUPAA coloproctology outpatient clinic. Faecal MRE analysis and subsequent evaluation of MRI spectra by statistical correlation (STOCSY) were performed by SIMCA software (umetrics.com). SRL was performed in the PubMed, Web of Science, Cochrane and Scopus databases, using key words related to colon cancer and MRE, considering studies published in English from 1992 to 2018. Results: CRC patients showed reduction in practically all metabolic pathways. Short chain fatty acids - SCFA (acetate, propionate and butyrate) and amino acids (alanine and glycine) were reduced against CG (ANOVA, P

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) O café é uma das bebidas mais populares no mundo e está dissertação apresenta o uso de diferentes métodos quimiométricos para análise de café torrado e moído. Em resumo, foram relatados métodos alternativos para análise de parâmetros de qualidade e de atributos sensoriais de amostras de café comercial pela espectroscopia no Infravermelho Próximo (NIR) e por Mínimos Quadrados Parciais (PLS). Adicionalmente, um modelo de discriminação para três qualidades de café foi proposto com sucesso utilizando a espectroscopia NIR combinada com os Mínimos Quadrados Parciais com Análise Discriminante (PLS-DA). Em ambos os casos, uma validação pela determinação dos parâmetros de mérito mostrou que todos os modelos de previsão apresentaram desempenho satisfatório. Além disso, uma análise metabolômica utilizando a espectroscopia de Ressonância Magnética Nuclear (1H-NMR) foi proposta. As informações metabolômicas obtidas pela 1H-NMR foram correlacionadas com os parâmetros de qualidade, atributos sensoriais incluindo a qualidade dos cafés e as informações espectrais do NIR utilizando o método ComDim. Os resultados mostraram que existe uma forte correlação entre as informações da espectroscopia de 1H-NMR com as propriedades sensoriais e as absorções no NIR e uma menor correlação com os parâmetros de qualidade. Em todas as aplicações desta dissertação, a combinação das técnicas de espectroscopia NIR e 1H-NMR em junção com os diferentes métodos quimiométricos utilizados, apresentaram-se como alternativas interessantes na análise do café torrado e moído. This dissertation presents the use of different chemometric methods for analysis of roasted and ground coffee. In summary, alternative methods for analyzing quality parameters and sensory attributes of commercial coffee by Near Infrared (NIR) Spectroscopy and by the Partial Least Squares (PLS) were reported. Additionally, a discrimination model for different coffees quality was successfully proposed using NIR spectroscopy combined with Partial Least Squares with Discriminant Analysis (PLS-DA). In both cases, a validation by the determination of the parameters of merit showed that all prediction models presented satisfactory performance. In addition, a metabolomic analysis using Nuclear Magnetic Resonance (1H-NMR) spectroscopy was proposed in two studies. In the first one, the information obtained by the 1H-NMR were correlated with the quality parameters, the sensory properties including the quality of the coffees and the spectral information of the NIR using the method ComDim. The results showed that there is a strong correlation between the information 1H-NMR spectroscopy with the sensory properties and the absorptions in the NIR and a lesser correlation with quality parameters. In all the applications of this dissertation, the combination of NIR and 1H-NMR spectroscopy techniques in conjunction with the different chemometric methods used, presented as interesting alternatives for analysis of roasted and ground coffee.

The use of bioactive glasses (e.g. silicates, phosphates, borates) has demonstrated to be an effective therapy for the restoration of bone fractures, wound healing and vascularization. Their partial dissolution towards the surrounding tissue has shown to trigger positive bioactive responses, without the necessity of using growth factors or cell therapy, which reduces money-costs, side effects and increases their translation to the clinics. However, bioactive glasses often need from stabilizers (e.g. SiO44−, Ti4+, Co2+, etc.) that are not highly abundant in the body and which metabolization is not fully understood. In this study, we were focused on synthesizing pure calcium phosphate glasses without the presence of such stabilizers. We combined a mixture of ethylphosphate and calcium 2-methoxyethoxide to synthesize nanoparticles with different compositions and degradability. Synthesis was followed by an in-depth nuclear magnetic resonance characterization, complemented with other techniques that helped us to correlate the chemical structure of the glasses with their physiochemical properties and reaction mechanism. After synthesis, the organically modified xerogel (i.e. calcium monoethylphosphate) was treated at 200 or 350 °C and its solubility was maintained and controlled due to the elimination of organics, increase of phosphate-calcium interactions and phosphate polycondensation. To the best of our knowledge, we are reporting the first sol-gel synthesis of binary (P2O5-CaO) calcium phosphate glass nanoparticles in terms of continuous polycondensated phosphate chains structure without the addition of extra ions. The main goal is to straightforward the synthesis, to get a safer metabolization and to modulate the bioactive ion release. Additionally, we shed light on the chemical structure, reaction mechanism and properties of calcium phosphate glasses with high calcium contents, which nowadays are poorly understood. Statement of Significance The use of bioactive inorganic materials (i.e. bioactive ceramics, glass-ceramics and glasses) for biomedical applications is attractive due to their good integration with the host tissue without the necessity of adding exogenous cells or growth factors. In particular, degradable calcium phosphate glasses are completely resorbable, avoiding the retention in the body of the highly stable silica network of silicate glasses, and inducing a more controllable degradability than bioactive ceramics. However, most calcium phosphate glasses include the presence of stabilizers (e.g. Ti4+, Na+, Co2+), which metabolization is not fully understood and complicates their synthesis. The development of binary calcium phosphate glasses with controlled degradability reduces these limitations, offering a simple and completely metabolizable material with higher transfer to the clinics.

The aim of this preliminary study was to evaluate meat quality properties, muscle metabolite profile and metabolic pathways associated with the occurrence of dark cutting meat in Angus x Nellore crossbreed cattle. After 14 days' ageing, dark cutting meat presented a higher pH, lower cooking loss and colour parameters, and greater tenderness compared with normal meat. Dark cutting meat had a higher ATP level and lower concentrations of glucose-6-phosphate, lactate, glucose, serine, threonine, creatine phosphate, inosine, leucine, methionine, succinate and glucose-1-phosphate compared to normal meat. In dark cutting samples, the ultimate pH was positively correlated with carnitine and negatively correlated with glucose-6-phosphate. However, in normal meat, the ultimate pH presented a positive correlation with arginine, leucine, methionine, proline, threonine, tyrosine and valine. Pathway analysis showed that differentiation of the groups was linked to energetic pathways such as starch and sucrose metabolism, the pentose phosphate pathway, amino sugar, nucleotide sugar metabolism, and glycolysis or gluconeogenesis. In conclusion, the occurrence of dark cutting meat has a notable impact on meat quality attributes and concentrations of post-mortem glycolytic metabolites, appears to be correlated with mitochondrial activity and affects energetic metabolic pathways.

Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of microsecond–millisecond motions opens the opportunity for regulating protein–protein interactions (PPIs) by modulating the dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active-site-directed inhibitors on the dynamics. We used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. We found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively abolished this equilibrium by stabilizing the enzyme in the closed conformation. This work was supported by the Institute for Research in Biomedicine, MINECO-FEDER (Bio2013-40716-R, CTQ2013-48287 and CTQ2012-32183/BQU), and the Generalitat de Catalunya (XRB and Grup Consolidat 2014SGR521). AL has received funding from the Instituto de Salud Carlos III. PB acknowledges the Agence Nationale de la Recherche (SPINHD-ANR-CHEX-2011) and the ATIP-Avenir program for financial support. FHT’s fellowship is co-funded by the INSERM and the University of Copenhagen. Technical assistance from staff at the P12 beam line (EMBL/DESY) is acknowledged.

Twenty-eight new Heusler phases REPd2Mg, REPd2Cd, REAg2Mg, REAu2Mg and REAu2Cd with different rare earth elements were synthesized from the elements in sealed niobium ampoules in a water-cooled sample chamber of an induction furnace. The samples were characterized by powder X-ray diffraction. The cell volumes show the expected lanthanide contraction. The structures of YPd2Cd, GdPd2Cd, GdAu2Cd, Y1.12Ag2Mg0.88 and GdAg2Mg were refined based on single crystal diffractometer data. The magnetic properties were determined for fifteen phase pure samples. LuAu2Mg is a weak Pauli paramagnet with a susceptibility of 1.0(2) × 10−5 emu mol−1 at room temperature. The remaining samples show stable trivalent rare earth ions and most of them order magnetically at low temperatures. The ferromagnet GdAg2Mg shows the highest ordering temperature of TC = 98.3 K. 113Cd and 89Y MAS NMR spectra of YAu2Cd and YPd2Cd confirm the presence of unique crystallographic sites. The resonances are characterized by large Knight shifts, whose magnitude can be correlated with electronegativity trends.

Background This study aimed to investigate associations between indicators of hypothalamic-pituitary-adrenal axis (HPA) functioning and metabolite levels in the anterior cingulate gyrus (ACG) of women with postpartum depression (PPD). Methods The sample (mean age = 28.5 ± 4.6 years) consisted of 20 women with PPD and 19 postpartum euthymic (PPE) women. Brain metabolites were quantified by proton magnetic resonance spectroscopy (1H-MRS). Salivary cortisol samples were collected upon awakening and 30 min and 12 h later, at 20.6 ± 6.6 (PPD) and 23.0 ± 7.4 (PPE) weeks after childbirth. Results There were no significant differences between groups in respect to metabolite levels in the ACG. Compared with PPE, PPD women had less diurnal variation (DVr%). In the PPD group, positive correlations were found between DVr% and myo-inositol (mI/Cr) levels, and between cortisol awakening response (CARi%) and glutamate + glutamine (Glx/Cr) levels. The correlation between CARi% and Glx/Cr remained significant even after controlling for the interval, in weeks, from birth and MR spectroscopy and to hormonal data collection, and the use of contraceptives. Limitations The limitations of the study include the small sample size and the use of oral contraceptives by around half of the sample. Conclusions In the remote postpartum period (mean 21.8 ± 6.9 weeks) and in the presence of depressive episodes, the decreased responsiveness of the HPA axis after awakening and a smaller decrease in cortisol levels over the day were associated with lower levels of metabolites in the ACG. These results may contribute to the development of biological models to explain the etiology of PPD.

[eng] Amyloid-beta peptide (Aβ) is strongly linked to the aetiology of Alzheimer’s disease (AD). Aβ is the main component of the amyloid plaques found in the brain of AD patients, however, Aβ is also present in the brain of healthy humans. It has been described that for this peptide to be neurotoxic, aggregation is needed. The accumulation of Aβ causes aggregation from low order oligomers through different intermediate species up to the formation of amyloid fibrils. However, is not the presence of the fibrils what correlates the harmfulness of the disease, but the concentration of soluble oligomeric intermediate species. Nowadays, is accepted that the neurotoxicity of these oligomers is produced on the membrane. From this point, the laboratory of Dr. Carulla developed the beta-barrel Pore Forming Oligomer (βPFO). βPFO, was produced with Aβ42, the most neurotoxic version of Aβ. It was the first described example of a stable, well-defined and homogeneous membrane oligomer with the ability to form pores on lipid membranes. In this thesis, we aim to advance in the characterization of this βPFO and the validation of βPFO as a druggable target for AD. First, as βPFO was described using detergents as a biomimetic membrane environment, we aimed to move towards a more native environment using natural lipids. By using lipid-detergent micelles we studied βPFO. In this work we demonstrated that βPFO is not able to reconstitute into the common 1,2-dihexanoyl-sn-glycero-3-phosphocholine - 1,2-dimyristoyl-sn-glycero-phosphatidylcholine (DHPC-DMPC) bicelles. Therefore, we described a new type of bicelles using dodecylphosphocholine (DPC) and DMPC, not described in the literature in these conditions until then. We showed that βPFO was able to reconstitute into DPC-DMPC bicelles preserving its overall structure and pore-forming function. Then, to advance towards βPFO validation, we immunized an alpaca with βPFO in order to generate Nanobodies. A Nanobody is a fragment of the single-chain antibodies produced by camelids, with many different properties from conventional antibodies, their reduced size, their cavity specificity, their ease of modification and production, etc. Upon the Nanobodies generation, we selected the ones specific against βPFO obtaining 11 different Nanobodies. Using enzyme-linked immunosorbent assay (ELISA) we showed that they had both, a high specificity for βPFO compared to monomeric and fibrillar Aβ42, and a high affinity for them. Moreover, we showed that these generated Nanobodies, were binding to βPFO in different manners affecting differently the protection they could cause to proteolysis. Finally, we demonstrated that upon Nanobody binding on membrane-inserted βPFO, some of the Nanobodies did not affect the current pass across the bilayer while others reduced the current pass and two of them completely blocked the pore formed. In the future, these Nanobodies could serve, not only as a tool to validate as a player βPFO in the context of AD, but also as possible therapeutics.

[eng] Nuclear Magnetic Resonance (NMR) spectroscopy is able to produce by a single direct measurement a very high amount of chemical information. However, this information is not always easy to interpret. In fact, the complexity of the NMR spectral data analysis is proportional to the number of compounds present simultaneously in the analyzed sample, as resonances from different compounds overlap. One of the most extreme situations can be found for NMR spectra of samples from metabolomics studies, from which approximately fifty compounds can be detected in a single measurement. In the study of the chemical processes involving metabolites (metabolomics), the most commonly used NMR spectra are the one-dimensional proton (1D 1H) NMR spectra, since they are relatively fast to acquire and proton sensitivity is the highest. The 1H-13C Heteronuclear Single Quantum Coherence (HSQC) NMR spectra are also frequently used in metabolomics for an improved structural characterization of the detected metabolites. In this Thesis, we have developed different data analysis strategies of 1H NMR and 1H-13C HSQC NMR metabolomics datasets. The investigated NMR spectra were acquired from extracts of Saccharomyces cerevisiae cells previously exposed to different environmental perturbations. The aim of these studies was to better understand the different metabolic processes that regulate the yeast metabolism acclimation to different growing conditions. From the study of these NMR metabolomics experiments, we designed new strategies and protocols for the analysis of these datasets that include the steps of data import, data pre-treatment, resonance assignment and metabolite quantification. Moreover, different chemometric methods were applied for the identification of the possible biomarkers that define the metabolic states of yeast cells and to extract the main metabolic profiles that describe the observed changes in the metabolome. Furthermore, two chemometric strategies were proposed for the untargeted analysis of 1H NMR and 1H-13C HSQC NMR, respectively. For the study of 1H NMR spectra of metabolomics samples, the application of the Multivariate Curve Resolution–Alternating Least Squares (MCR-ALS) chemometric method allowed the satisfactory resolution of the individual 1H NMR spectra and concentrations of the different metabolites. On the other hand, the investigation of metabolomics datasets by 1H-13C HSQC NMR revealed that most of the data values in these NMR spectra are only descriptive of noise, hampering their chemometric data analysis. In this context, a new strategy to filter the variables relative to noise, named ‘Variables of Interest’ (or VOI) is proposed. After the application of this procedure, we observed that the analysis of the noise-filtered 1H-13C HSQC NMR spectra produced similar results to the corresponding analysis of 1H NMR spectra. Due to the existence of the second dimension in the 1H-13C HSQC NMR spectra, resonances are less overlapped and they could be integrated without using deconvolution approaches. For all these reasons, and linked to the fact that more chemical information is contained in the 1H-13C HSQC NMR spectra than in the 1H NMR spectra, the analysis of noise-filtered 1H-13C HSQC NMR spectra allow a more accurate characterization of the metabolomic system, in a reduced amount of time in comparison to the analysis of the corresponding 1H NMR spectra., [cat] L'espectroscòpia de ressonància magnètica nuclear (RMN) és capaç de generar mitjançant una mesura simple i directa una gran quantitat d'informació química. Tanmateix, aquesta informació no sempre és fàcil d'interpretar. De fet, la complexitat de l'anàlisi espectral és proporcional al nombre de compostos presents en la mostra analitzada, ja que les ressonàncies dels diferents compostos es troben superposades. Una de les situacions més extremes la podem trobar en el cas dels espectres de RMN de mostres obtingudes en estudis de metabolòmica, en les que es poden arribar a detectar al voltant d’una cinquantena de compostos en una sola mesura. En l'estudi dels processos químics relacionats amb els metabòlits (metabolòmica), els espectres de RMN més utilitzats són els espectres monodimensionals de protó (1D 1H), ja que són relativament ràpids d'adquirir i la sensibilitat del protó és la més alta. És també corrent utilitzar en estudis de metabolòmica els espectres de RMN bidimensionals 1H-13C heteronuclears de coherència quàntica única (2D 1H-13C HSQC), els quals permeten obtenir una millor caracterització estructural dels metabòlits detectats. En aquesta Tesi, s’han desenvolupat diferents estratègies d'anàlisi d’espectres de RMN de 1H i de 1H-13C HSQC de mostres de metabolòmica. Els espectres de RMN van ser adquirits d’extractes de llevat Saccharomyces cerevisiae que prèviament havia estat exposat a diferents pertorbacions mediambientals. L’objectiu d’aquests estudis ha estat millorar la comprensió dels diferents processos metabòlics que regulen l'aclimatació de les cèl·lules de llevat a diferents condicions de creixement. A partir d’aquests estudis de metabolòmica realitzats, es van dissenyar noves estratègies i protocols d'anàlisi de dades de RMN que inclouen la seva importació, el seu preprocessament, l'assignació de les ressonàncies i la seva integració. A més, es van aplicar diferents mètodes quimiomètrics que van permetre identificar els biomarcadors de l’estat metabòlic de les cèl·lules del llevat i extreure els principals perfils metabòlics que descriuen els canvis en el seu metabolisme. Es van proposar a més, dues estratègies quimiomètriques per a l’anàlisi no dirigida d’espectres de RMN de 1H i de 1H-13C HSQC, respectivament. En el cas dels estudis d’espectres de RMN de 1H, l'aplicació del mètode de resolució multivariant de corbes per mínims quadrats alternats (MCR-ALS) va permetre resoldre satisfactòriament les concentracions i els espectres individuals dels diferents metabòlits. D’altra banda, la investigació de l’estructura de les dades dels espectres de RMN de 1H-13C HSQC va revelar que la majoria dels valors espectrals són descriptius del soroll, cosa que dificulta la seva anàlisi. En aquest context, s’ha desenvolupat una nova estratègia per filtrar les variables descriptives del soroll, anomenada selecció de les variables d'interès (Variables of Interest, VOI). Després d’aplicar aquest procediment, es va observar que l'anàlisi dels espectres 1H-13C HSQC filtrats produeix resultats similars als obtinguts amb els espectres corresponents de 1H. Degut a l’existència de la segona dimensió en els espectres de 1H-13C HSQC, les ressonàncies estan menys solapades i es poden integrar sense fer servir estratègies basades en la seva deconvolució. Degut a tot això i al fet que els espectres de 1H-13C HSQC contenen més informació química que els de 1H, l’anàlisi dels espectres de 1H-13C HSQC filtrats amb aquest procediment permet una caracterització del sistema metabolòmic més acurada i amb temps d’anàlisis més curts, en comparació a l’anàlisi dels espectres de 1H corresponents.

We present Farseer-NMR (https://git.io/vAueU), a software package to treat, evaluate and combine NMR spectroscopic data from sets of protein-derived peaklists covering a range of experimental conditions. The combined advances in NMR and molecular biology enable the study of complex biomolecular systems such as flexible proteins or large multibody complexes, which display a strong and functionally relevant response to their environmental conditions, e.g. the presence of ligands, site-directed mutations, post translational modifications, molecular crowders or the chemical composition of the solution. These advances have created a growing need to analyse those systems’ responses to multiple variables. The combined analysis of NMR peaklists from large and multivariable datasets has become a new bottleneck in the NMR analysis pipeline, whereby information-rich NMR-derived parameters have to be manually generated, which can be tedious, repetitive and prone to human error, or even unfeasible for very large datasets. There is a persistent gap in the development and distribution of software focused on peaklist treatment, analysis and representation, and specifically able to handle large multivariable datasets, which are becoming more commonplace. In this regard, Farseer-NMR aims to close this longstanding gap in the automated NMR user pipeline and, altogether, reduce the time burden of analysis of large sets of peaklists from days/weeks to seconds/minutes. We have implemented some of the most common, as well as new, routines for calculation of NMR parameters and several publication-quality plotting templates to improve NMR data representation. Farseer-NMR has been written entirely in Python and its modular code base enables facile extension. Electronic supplementary material The online version of this article (10.1007/s10858-018-0182-5) contains supplementary material, which is available to authorized users.

Dissolution dynamic nuclear polarization (DNP) has become one of the predominant implementations for DNP. However, the technical implementation of transferring the sample from the polarizer to the nuclear magnetic resonance (NMR) system remains challenging. There is a need for additional technical optimizations in order to use dissolution DNP for biochemical and chemical applications. Here we show how a newly designed pressure dissolution kit considerably improves spectral quality and stability by enabling highly reliable and fast sample transfer to the NMR system.

S.P.N. thanks the ERC (Advanced Investigator Award ‘FUNCAT’), the EPSRC and Syngenta for funding. S.P.N. is a Royal Society Wolfson Merit Award Holder. D.J.N. thanks the University of Strathclyde for a Chancellor's Fellowship. A.P. thanks the Spanish MINECO for a Ramón y Cajal contract (RYC-2009-05226) and the European Commission for a Career Integration Grant (CIG09-GA-2011-293900). The electronic nature of the interaction of NHCs with metal centres is of interest when exploring their properties, how these properties influence those of metal complexes, and how these properties might depend on ligand structure. Selenourea and phosphinidene complexes have been proposed to allow the measurement of the π-accepting ability of NHCs, independent of their σ-donating ability, via the collection of 77Se or 31P NMR spectra, respectively. Herein, the synthesis and characterisation of selenoureas derived from a range of imidazol-2-ylidenes, 4,5-dihydroimidazol-2-ylidenes and triazol-2-ylidenes are documented. Computational studies are used to explore the link between the shielding of the selenium centre and the electronic properties of the NHCs. Results show that δSe is correlated to the energy gap between a filled lone pair orbital on Se and the empty π* orbital corresponding to the Se–NHC bond. Bond energy decomposition analysis indicated no correlation between the orbital σ-contribution to bonding and the chemical shielding, while a good correlation was found between the π-contribution to bonding and the chemical shielding, confirming that this technique is indeed able to quantify the ability of NHCs to accept π-electron density. Calculations conducted on phosphinidene adducts yielded similar results. With the link between δSe and δP and π-back bonding ability clearly established, these compounds represent useful ways in which to fully understand and quantify this aspect of the electronic properties of NHCs. Publisher PDF

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Esta tese de doutorado aborda o preparo, a caracterização e a utilização de fases estacionárias monolíticas para uso em colunas de separação cromatográfica em escala capilar. As fases estacionárias foram preparadas pela polimerização do monômero 3-(metacriloxipropil)-trimetoxisilano em duas etapas envolvendo primeiramente um processo sol-gel e depois um processo de adição radicalar fotoiniciado. A fotopolimerização foi feita in situ em capilares de sílica fundida transparentes à radiação UV com 100 μm de diâmetro interno. As colunas produzidas foram aplicadas qualitativamente com sucesso na separação de cinco hidrocarbonetos policíclicos aromáticos e três hormônios esteroides, tanto por cromatografia a líquido capilar quanto por eletrocromatografia capilar em um equipamento de eletroforese capilar. A eletrocromatografia foi usada para analisar quantitativamente uma amostra de emulsão transdérmica contendo estriol e 17-β-estradiol. Como tentativa de melhorar a separação entre alguns analitos, foi feita a modificação do polímero adicionando o monômero benzil metacrilato, porém sem sucesso. As colunas foram caracterizadas com relação à sua homogeneidade física usando um microscópio óptico e um detector condutométrico sem contato capacitivamente acoplado. O detector foi usado como um scanner não invasivo deslocando-se longitudinalmente ao longo da coluna externamente ao capilar. A caracterização química do polímero foi feita através da espectroscopia de ressonância magnética nuclear no estado sólido dos núcleos de 13C e de 29Si. Foi possível propor uma estrutura para o arranjo da cadeia carbônica e o grau de substituição do grupo silil indicando a reticulação das cadeias poliméricas através da formação de dímeros e trímeros condensação de grupos alcoxisilanos. This doctoral thesis adresses the preparation, characterization and use of monolithic stationary phases for chromatographic separation columns in capillary scale. The stationary phases were prepared by polymerizing the 3- (methacryloxypropyl) trimethoxysilane monomer in two steps involving a sol-gel process followed by a photoinitiated radical addition. The photopolymerization was carried out in UV transparent coating fused silica capillaries with 100 μm of internal diameter. The columns were applied in the qualitative separation of five polycyclic aromatic hydrocarbons and three steroid hormones, both by capillary liquid chromatography and capillary electrochromatography in a capillary electrophoresis equipment. Electrochromatography was used to quantitative analyzis of a transdermal emulsion sample containing estriol and 17-β-estradiol. In an attempt to improve the separation between some analytes, the polymer was modified by adding the benzyl methacrylate monomer, but without success. The columns were characterized with respect to their physical homogeneity using an optical microscope and a capacitively coupled contactless conductivity detector. The detector was used as a non-invasive scanner moving longitudinally along the column externally to the capillary. The chemical characterization of the polymer was done by the solid state 13C and 29Si nuclear magnetic resonance spectroscopy. It was possible to propose a carbon chain strucuture and the substitution degree of the silyl group indicating the cross-linking of the polymer chains through the formation of dimers and trimers by condensation of the alkoxysilane groups.

[eng] The Escherichia coli infecting T7 bacteriophage shares a common dsDNA packaging mechanism with other bacteriophages of the Caudovirales order, Herpesviruses and Adenoviruses. The packaging machinery comprises the portal protein and the terminase complex. The portal protein is a channel located at a unique portal vertex that provides a conduit for DNA translocation, while the terminase complex recognizes a long concatemer of DNA, performs the nuclease catalytic activity and hydrolyses ATP. Available structural information about portal proteins describes them as oligomeric rings with an axial channel. High quality samples suitable for structural characterization of the portal protein of T7 bacteriophage were obtained and characterized. Both X-ray crystallography and cryo-electron microscopy (cryo-EM) data were collected, and an initial model built on the 5.8Å cryo-EM map was used to phase the crystallographic data, which allowed the building of a model of a tridecameric particle at 2.8Å resolution. The T7 portal particle is 170Å tall and 110Å wide toroidal protein with a central channel that ranges from 23Å to 95Å in diameter. Four domains have been identified in the structure: the wing, the stem, the clip and the crown. The a10-tunnel loop valve is proposed to play an important functional role. During packaging, it may adapt while DNA is translocated and rotated, and once the genome has been packed the side chain of tunnel loop residue Arg368 may be able to seal the channel and stabilize the DNA inside the capsid before tail assembly. Interestingly, these mechanisms would not only imply the flexibility of a loop region, but also the kink of the longer helix of the portal structure, a10.

The study of biomarkers of dietary patterns including the Mediterranean diet (MedDiet) is scarce and could improve the assessment of these patterns. Moreover, it could provide a better understanding of health benefits of dietary patterns in nutritional epidemiology. We aimed to determine a robust and accurate biomarker associated with a high adherence to a MedDiet pattern that included dietary assessment and its biological effect. In this cross-sectional study, we included 56 and 63 individuals with high (H-MDA) and low (L-MDA) MedDiet adherence categories, respectively, all from the Prevencion con Dieta Mediterranea trial. A 1H-NMR-based untargeted metabolomics approach was applied to urine samples. Multivariate statistical analyses were conducted to determine the metabolite differences between groups. A stepwise logistic regression and receiver operating characteristic curves were used to build and evaluate the prediction model for H-MDA. Thirty-four metabolites were identified as discriminant between H-MDA and L-MDA. The fingerprint associated with H-MDA included higher excretion of proline betaine and phenylacetylglutamine, among others, and decreased amounts of metabolites related to glucose metabolism. Three microbial metabolites - phenylacetylglutamine, p-cresol and 4-hydroxyphenylacetate - were included in the prediction model of H-MDA (95% specificity, 95% sensitivity and 97% area under the curve). The model composed of microbial metabolites was the biomarker that defined high adherence to a Mediterranean dietary pattern. The overall metabolite profiling identified reflects the metabolic modulation produced by H-MDA. The proposed biomarker may be a better tool for assessing and aiding nutritional epidemiology in future associations between H-MDA and the prevention or amelioration of chronic diseases.

Doutoramento em Nanociências e Nanotecnologia Face ao uso disseminado e enorme potencial terapêutico das nanopartículas de prata (AgNPs), o estudo dos seus efeitos biológicos é um assunto relevante e atual. O trabalho apresentado nesta tese teve como objetivo aprofundar o conhecimento existente sobre o impacto das AgNPs no metabolismo celular, usando a metabolómica por espectroscopia de ressonância magnética nuclear (RMN). Os tipos celulares escolhidos para este estudo foram queratinócitos da epiderme humana, células de hepatoma humano e macrófagos sanguíneos, por serem relevantes, respetivamente, ao nível da entrada, acumulação e captação de nanopartículas no organismo. O Capítulo 1 introduz as principais propriedades das AgNPs, a sua atividade biológica e potencial toxicidade, e descreve a abordagem metabolómica, incluindo uma breve revisão bibliográfica das suas aplicações em nanotoxicologia. O âmbito e os objetivos desta tese são, também, apresentados. O Capítulo 2 descreve os métodos experimentais utilizados ao longo deste trabalho, incluindo a caracterização das AgNPs, os procedimentos usados na cultura celular e nos ensaios biológicos, a colheita e preparação de amostras, a análise por RMN e o tratamento estatístico dos dados. No Capítulo 3, a atividade metabólica e a composição dos três tipos de células usados neste trabalho (queratinócitos HaCaT, células de hepatoma HepG2 e macrófagos RAW 264.7) são descritas com base na análise por RMN dos sobrenadantes dos meios de cultura (exometaboloma) e dos extratos celulares polares e lipofílicos (endometaboloma). O Capítulo 4 apresenta a análise metabolómica das células HaCaT expostas a AgNPs de diferentes tamanhos (10, 30 ou 60 nm de diâmetro) e revestimentos (citrato, polietilenoglicol ou albumina de soro bovino). Verificou-se que o metaboloma celular foi afetado mesmo a concentrações subtóxicas de AgNPs, sugerindo: aumento da glicólise e glutaminólise, alteração na atividade do ciclo dos ácidos tricarboxílicos (TCA) e nos processos de produção e transferência de energia, degradação de proteínas, síntese de glutationa (GSH), modificações a nível das membranas e do equilíbrio osmótico. Apesar de muitas variações serem comuns a todas as nanopartículas testadas, as AgNPs de 10 nm causaram os efeitos mais distintos, nomeadamente no que diz respeito à glicólise e à síntese/utilização de GSH. Além disso, a exposição celular a prata iónica (Ag+) confirmou o importante papel dos iões prata no mecanismo de ação das AgNPs, enquanto a comparação com o peróxido de hidrogénio (H2O2) permitiu destacar os efeitos relacionados com o stress oxidativo. No Capítulo 5 são apresentadas as respostas metabólicas das células de fígado HepG2 a dois tipos de AgNPs, umas obtidas por redução química e estabilizadas em citrato e as outras obtidas por síntese verde na presença de um extrato vegetal (Cit30 e GS30, respetivamente), ambas com centros metálicos de 30 nm. Os resultados sugeriram adaptações metabólicas em processos de produção de energia (metabolismo da glucose e sistema da fosfocreatina), autofagia e metabolismo lipídico, refletindo possivelmente a ativação de mecanismos de proteção. Ainda que os dois tipos de AgNPs tenham induzido muitos efeitos semelhantes, as Cit30 pareceram causar um maior impacto no ciclo TCA e na degradação de proteínas, enquanto as GS30 aparentaram induzir uma diminuição mais forte na síntese de fosfolípidos. A assinatura metabólica da prata iónica foi bastante semelhante à das AgNPs, sugerindo, no entanto, uma menor capacidade das células expostas a Ag+ extracelular para lidar com o stress oxidativo. O Capítulo 6 descreve a avaliação do impacto das AgNPs Cit30 no metaboloma de macrófagos de murganho RAW 264.7, a concentrações subtóxicas (decréscimos de ~5 e 20% na viabilidade celular). As alterações encontradas apontaram para: estimulação da glicólise (a baixa concentração de exposição), reprogramação do ciclo TCA (resultando numa intensa produção de itaconato e succinato e numa marcada depleção de ATP, consistentes com uma resposta pro-inflamatória), ativação da gluconeogénese, promoção da síntese de GSH e acumulação de creatina/fosfocreatina. Foram, ainda, observadas variações possivelmente relacionadas com a osmorregulação e a modificação membranar. De notar que os macrófagos expostos a Ag+ mostraram características semelhantes aos expostos a AgNPs (por ex., aumento da glucose intracelular – gluconeogénese), mas também revelaram efeitos distintos, nomeadamente em metabolitos envolvidos no ciclo TCA, em processos de transferência de energia e no metabolismo lipídico. Adicionalmente, viu-se que o metaboloma dos macrófagos respondeu de maneira diferente à exposição a H2O2 (por ex., tendência para diminuição da glicólise e sem efeitos observados na ativação da gluconeogénese ou da síntese de GSH), indicando que muitos dos efeitos induzidos pelas AgNPs não foram necessariamente mediados por stress oxidativo. Finalmente, com base na integração dos resultados apresentados ao longo dos capítulos anteriores, as principais conclusões deste trabalho são apresentadas e discutidas no Capítulo 7. The wide dissemination and promising therapeutic potential of silver nanoparticles (AgNPs) make the study of their biological effects a relevant upto- date subject. The work presented in this thesis aimed at deepening current understanding of the impact of AgNPs on cell metabolism, using NMR metabolomics of cultured mammalian cells. Epidermis keratinocytes, hepatoma cells and blood macrophages, relevant, respectively, to nanoparticle entry, accumulation and uptake, have been selected for the study. Chapter 1 introduces the main properties of AgNPs, including their biological activity and toxicological potential, and describes the metabolomics approach, briefly reviewing its applications in the field of nanotoxicology. Also, the scope and aims of this thesis are presented. Chapter 2 covers the experimental methods adopted during the course of this work, including the sources and characterisation of AgNPs, the procedures used in cell culture and biological assays, sample collection and preparation methods, NMR analyses and statistical data treatment. In Chapter 3, the metabolic activity and composition of the three cell types used in this work (HaCaT keratinocytes, HepG2 hepatoma cells and RAW 264.7 macrophages) are described based on the NMR analysis of culture medium supernatants (exometabolome) and polar/lipophilic cell extracts (endometabolome). Chapter 4 presents the metabolomic analysis of HaCaT skin cells exposed to AgNPs of different sizes (10, 30 or 60 nm in diameter) and coatings (citrate, polyethylene glycol or bovine serum albumin). The cellular metabolome was found to be affected even at sub-toxic concentrations of AgNPs, suggesting: upregulation of glycolysis and glutaminolysis, altered tricarboxylic acid (TCA) cycle activity, protein degradation, disruption of energy-producing pathways, glutathione (GSH) synthesis, membrane modification and changes in osmotic balance. Although several metabolic variations were common to all tested nanoparticles, the 10 nm AgNPs showed the most distinct effects, namely in regard to glycolysis and GSH synthesis/utilisation. Furthermore, cell exposure to ionic silver (Ag+) confirmed the major role of silver ions in AgNPs mode of action, while comparison with hydrogen peroxide (H2O2) allowed the effects related to oxidative stress to be highlighted. In Chapter 5, the metabolic responses of liver HepG2 cells to two types of 30 nm AgNPs, obtained by chemical reduction and stabilised with citrate or produced by green synthesis in the presence of a plant extract (Cit30 and GS30, respectively) are presented. Sub-toxic concentrations of AgNPs were proposed to induce metabolic adaptations in energy production processes (glucose metabolism and the phosphocreatine system), autophagy and lipid metabolism, possibly reflecting the activation of metabolism-mediated protective mechanisms. Although the two types of AgNPs induced many common effects, Cit30 appeared to have a greater impact on the TCA cycle and protein degradation, whereas GS30 seemed to induce stronger downregulation of phospholipid synthesis. The metabolic signature of Ag+ was largely similar to that of AgNPs, although suggesting a lower ability of cells exposed to extracellular Ag+ to cope with oxidative stress. Chapter 6 addresses the impact of Cit30 AgNPs on the metabolome of murine RAW 264.7 macrophages, at concentrations causing minimal (~5 and 20%) decreases in cell viability. Exposed cells were suggested to upregulate glycolysis (at the low exposure concentration), to reprogram the TCA cycle (resulting in marked production of itaconate and succinate and in ATP depletion, consistent with a pro-inflammatory response), to activate gluconeogenesis, to promote GSH synthesis, and to increase the creatine/phosphocreatine pool. Changes putatively related to osmoregulation and membrane modification were also observed. Notably, macrophages exposed to Ag+ showed common features to AgNPs-exposed cells (e.g. increased intracellular glucose suggesting gluconeogenesis), but also several distinct effects, for instance in metabolites involved in the TCA cycle, energy transfer processes or lipid metabolism. Furthermore, the cellular metabolome responded differently to H2O2 exposure (e.g. trend for downregulated glycolysis, no evidence of gluconeogenesis activation or of GSH upregulation), indicating that many of the AgNPs-induced effects were not necessarily mediated by oxidative stress. Finally, based on the integration of the results presented along the previous chapters, the main conclusions of this work are presented and discussed in Chapter 7.

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