O processo de extração do óleo de mamona gera, como subproduto, uma torta rica em proteínas, que tem sido utilizada como adubo e contra fitonematóide, devido à sua toxicidade. Entretanto, as proteínas da torta de mamona são ricas em aminoácidos que permitem modificação química com aldeídos bifuncionais, como o glutaraldeído e o glioxal, o que pode ser interessante para a produção de filmes biodegradáveis com propriedades físicas adequadas ao emprego na agricultura. Assim, o objetivo geral deste trabalho foi a produção e caracterização de filmes à base de proteínas extraídas da torta de mamona e reticuladas com glioxal ou glutaraldeído e plastificados com glicerol. Especificamente, estudaram-se o efeito da concentração de proteína e do tipo de reticulante, e o efeito da concentração do reticulante sobre algumas propriedades dos filmes. Os filmes foram produzidos com uma técnica conhecida como tipo \"casting\", que consiste na desidratação de uma solução filmogênica (SF). Os filmes foram submetidos a testes para determinação da umidade, espessura, propriedades mecânicas (testes de tração e perfuração), permeabilidade ao vapor de água, solubilidade em água, cor, opacidade, brilho, além da microestrutura, por microscopia eletrônica de varredura. A concentração da proteína e o tipo de reticulante não influenciaram a umidade, espessura, microestrutura e propriedades óticas. Mas, de maneira geral, os filmes produzidos com o glioxal apresentaram melhores propriedades mecânicas e menor solubilidade em água que aqueles produzidos com glutaraldeído, sendo que a concentração de 6g de proteínas/100g de SF foi a formulação que apresentou os melhores resultados para os testes de tração e de solubilidade em água. Este resultado é muito importante para o emprego dos filmes na agricultura. Por outro lado, a concentração do glioxal não teve efeito sobre a umidade, espessura, propriedades óticas, microestrutura e nem sobre a permeabilidade ao vapor de água, influenciando apenas a solubilidade e as propriedades mecânicas. O teor de glioxal que proporcionou melhores propriedades mecânicas e baixa solubilidade em água foi de 5g/100g de proteínas. Filmes produzidos nessas melhores condições foram submetidos a análises complementares envolvendo testes de hidrofobicidade, propriedades térmicas, espectroscopia de infravermelho com transformada de Fourier, isoterma de sorção e biodegradabilidade. Finalmente, pode-se concluir que as proteínas da torta de mamona são capazes de formar uma matriz bem estruturada e que suas propriedades podem ser melhoradas com a adição de glioxal. / The extraction process of castor oil produces, as a co-product, a cake rich in proteins which has been used as a fertilizer and against plant parasitic nematode, because of its toxicity. However, the proteins of castor bean cake are rich in amino acids which allow chemical modification with bifunctional aldehydes such as glutaraldehyde and glioxal. This may be interesting for the production of biodegradable films with adequate physical properties to the use in agriculture. The objective of this work was the production and characterization of films based on proteins extracted from the castor bean cake and crosslinked with glyoxal or glutaraldehyde and plasticized with glycerol. Specifically, we studied the effect of protein concentration and type of crosslinker and the effect of the crosslinker concentration on some properties of the films. The films were produced with a technique known as \"casting\", which consists of drying a film solution (FS). The films were tested for determination of moisture content, thickness, mechanical properties (tensile and puncture tests), water vapor permeability, water solubility, color, opacity, gloss, and the microstructure by scanning electron microscopy. The protein concentration and type of crosslinking did not affect the moisture content, thickness, microstructure and optical properties. But, in general, films made with glyoxal showed improved mechanical properties and lower water solubility than those produced using glutaraldehyde. And, the concentration of 6g proteins/100 g of FS was the formulation that presented the best results for the tensile tests and water solubility. This result is very important for the use of films in agriculture. Moreover, the concentration of glyoxal had no effect on the moisture content, thickness, optical properties, or on the microstructure and permeability to water vapor, influencing only the solubility and mechanical properties. The concentration of glyoxal that provided the best mechanical properties and low water solubility of proteins was 5g/100g of gelatin. Films produced in the best conditions were subjected to further analyzes involving tests of hydrophobicity, thermal properties, spectroscopy, Fourier transform infrared spectroscopy, water vapor sorption isotherm and biodegradability. Finally, it can be concluded that the protein of castor bean cake are capable of forming a matrix structured and that their properties can be improved with the addition of glyoxal.

De nombreuses études dans la seconde moitié du 20ème siècle, ont mis en évidence que des génotoxiques cancérogènes réagissent avec l'ADN pour former par liaison covalente des adduits qui sont impliqués dans le processus cancérigène. Bien qu’il existe des preuves convaincantes de la présence de multiples adduits à l'ADN dans les poumons de sujets exposés au tabagisme ou en milieu professionnel à un aldéhyde donné, il est évident que c'est un domaine dans lequel des recherches supplémentaires ont été nécessaires. L’objectif de ce travail de thèse est d’établir des profils d’adduits exocycliques à l'ADN induits par le mélange d’aldéhydes, qui pourraient à terme être considérés comme un marqueur génotoxique de l’exposition aux aldéhydes, tant endogène qu’environnemental. Pour cette raison, nous avons validé une méthode en UHPLC-MS/MS rapide, sensible et précise en utilisant la dilution isotopique, pour la quantification à l’état de trace de 9 adduits exocycliques à l’ADN dérivés de 8 principaux aldéhydes exogènes et endogènes, notamment le formaldéhyde, l’acétaldéhyde, l’acroléine, le crotonaldéhyde, le malondialdéhyde, le 4-hydroxy-2-nonénal, le glyoxal et le méthylglyoxal. Ces adduits ont été synthétisés et purifiés ainsi que leurs homologues marqués au 13C10, 15N5, identifiés et quantifiés par le biais des courbes d'étalonnage allant de 0,25 à 250 ng/mL d'adduits dans l'eau et l'ADN afin de décrire les effets matrice. Des échantillons de contrôle qualité ont été préparés et analysés afin de vérifier l'exactitude et la précision de la méthode dans des situations de répétabilité et de fidélité intermédiaire. L'absence de contamination croisée a également été démontrée. La méthode est capable de différencier les 9 analytes d'intérêt et leurs étalons internes en utilisant pour chaque analyte une transition de quantification et une seconde de confirmation. Cette méthode a été validée selon les recommandations de l'Agence Européenne des Médicaments concernant les méthodes bioanalytiques. Elle répond à tous les critères essentiels pour garantir l'acceptabilité des performances et la fiabilité des résultats d'analyse. Cette méthode est la toute première validée et peut être utilisée en adductomique dans le cadre d'études sur l'exposome. En plus, nous avons simultanément mesuré par une approche in vitro les 9 adduits exocycliques dans de l’ADN de thymus de veau exposé à de différentes concentrations de chaque aldéhyde seul ou en mélanges équimolaires. Cette approche nous a permis d’établir des relations dose-dépendantes pour tous les aldéhydes à l’exception du malondialdéhyde et du méthylglyoxal. Une relation dose-réponse a également été observée avec les mélanges équimolaires d’aldéhydes. Elle a permis de définir des réactivités différentes des aldéhydes en mélange vis-à-vis de l’ADN. Les profils de ces adduits exocycliques ont été également déterminés dans l'ADN de sang de fumeurs et de non-fumeurs. La fumée de cigarette contient plusieurs aldéhydes connus de se lier par covalence aux bases de l’ADN, ainsi l’adduit à l’ADN peut être considéré comme biomarqueur d’exposition au tabac. Des différences significatives dans les niveaux d’adduits ont été obtenues entre l’ADN des fumeurs et celui des non-fumeurs à l’exception de l’adduit induit par le malondialdéhyde. Des corrélations ont été établies entre chaque adduit et les marqueurs de la consommation tabagique sans aucune corrélation significative de la totalité des adduits avec un marqueur spécifique. Par ailleurs, nous avons montré que l’exposition au formaldéhyde, au butanal et au benzaldéhyde a eu un effet sur les concentrations du MDA urinaire mesurées chez les policiers libanais stationnés au carrefour pendant 7 h par jour et après exposition de 5 jours aux émissions du trafic routier. Une augmentation du MDA plasmatique a été décrite ; les années de travail avaient une incidence sur les concentrations de ce biomarqueur. / Many studies in the second half of the 20th century have shown that genotoxic carcinogens, either directly or after metabolic activation, react with DNA to form covalently bonded adducts that are absolutely central in the carcinogenic process. Although there is compelling evidence of the presence of multiple DNA adducts in the lungs of subjects exposed to smoking or occupational exposure to a given aldehyde, it is clear that this is an area in which further research has been necessary. The aim of this thesis is to establish exocyclic DNA adducts profiles induced by the mixture of aldehydes, which could eventually be considered as a genotoxic marker of aldehyde exposure, both endogenous environmental. For this reason, we have validated a fast, sensitive and precise method on liquid chromatography coupled to mass spectrometry in tandem mode (UHPLC-MS/MS) using isotopic dilution, for trace quantification of 9 exocyclic DNA adducts derived from 8 major exogenous and endogenous aldehydes, including formaldehyde, acetaldehyde, acrolein, crotonaldehyde, malondialdehyde, 4-hydroxy-2-nonenal, glyoxal and methylglyoxal. These adducts were synthesized and purified as well as their labeled homologues, identified and quantified through standard curves ranging from 0.25 (LLOQ) to 250 ng/mL (ULOQ) adducts in water and in DNA to describe the matrix effects. Quality control (QC) samples were prepared and analyzed to verify the accuracy and precision of the method in repeatability and intermediate fidelity situations. The absence of cross-contamination has also been demonstrated. The method is able to differentiate the 9 analytes of interest and their internal standards using for each analyte a quantification transition and a confirmation transition. This method has been validated according to the recommendations of the European Medicines Agency (EMA) concerning bioanalytical methods. It meets all the essential criteria to guarantee the acceptability of the performances and the reliability of the analysis results. This method is the very first validated and can be used in adductomics in the context of studies on the exposome. In addition, the exocyclic adducts were simultaneously measured by an in vitro approach in calf thymus DNA exposed to different concentrations of each aldehyde apart or in equimolar mixtures. This approach allowed us to establish dose-dependent relationships for all aldehydes with the exception of malondialdehyde and methylglyoxal. A dose-response relationship was also observed with equimolar mixtures of aldehydes. It made it possible to define different reactivities of aldehydes in mixture versus DNA. The profiles of these exocyclic adducts were also determined in the blood DNA of smokers and non-smokers. Cigarette smoke contains several aldehydes known to covalently bind to DNA bases, so the DNA adduct may be considered as biomarker of tobacco exposure. Significant differences in adducts levels were obtained between smokers and non-smokers DNA with the exception of malondialdehyde-induced DNA adduct. Correlations were established between each adduct and smoking-related markers without any significant correlation of all adducts with a specific marker. Furthermore, we have shown that exposure to formaldehyde, butanal and benzaldehyde had an effect on the concentrations of urinary MDA measured in Lebanese police stationed at the intersection for 7 hours a day and after 5-day exposure to road traffic. An increase in plasma MDA has been described; years of work had an impact on the concentrations of this biomarker. These results are promising and it would be interesting to validate in population the profile of 9 exocyclic adducts as biomarkers of exposure to both exogenous and endogenous aldehydes as part of an adductomic approach to understand the carcinogenic risk in relation to aldehydes exposures in urban areas.

The nucleophilic addition of organomagnesium/lithium reagents to aldehydes and ketones has long enabled the synthesis of valuable alcohol derivatives; however, these types of transformations are often plagued by poor functional group tolerance and require harsh reaction conditions. The direct coupling of carbonyls and alcohols with aryl halides is an appealing alternative to access secondary alcohol products. However, this necessitates a formal C-H bond activation which is not well established in the literature. Chapter 1 provides a detailed literature background of the transition metal-catalyzed functionalization of carbonyls and alcohols. The work discussed in Chapter 2 of this thesis demonstrates the addition of aryl halides to aryl and aliphatic aldehydes and alcohols providing secondary alcohol products in moderate to high yields. Key to the success of this transformation was the implementation of underexplored and readily synthesized 1,5-diaza-3,7-diphosphacyclooctane (P2N2) ligands. Chapter 3 extends the methodology established in chapter 2 and aims to get a preliminary understanding of the application and mechanism of the reaction described above. For this purpose, pharmaceutically relevant isatin substrates are derivatized, providing access to substitution at the 3-position. Coupling isatins with aryl halides yields 3-aryl-3-hydroxy-2-oxindole products which are scaffolds for many natural product derivatives. Through high-throughput experimentation (HTE), we were able to discover that 1,2-addition at the carbonyl position of isatins is highly compatible with our established system and led us to develop a modest scope as well as gain useful mechanistic insights for this coupling.

No description available.

Chiral molecules play a central role in our daily life and in nature, for instance the different enantiomers or diastereomers of a chiral molecule may show completely different biological activity. For this reason, it is a vital goal for synthetic chemists to design selective and efficient methodologies that allow the synthesis of the desired enantiomer. In this context, it is highly important that the concept of green chemistry is considered while designing new approaches that eventually will provide more environmental and sustainable chemical synthesis.The aim of this thesis is to develop the concept of combining transition metal catalysis and aminocatalysis in one process (dual catalysis). This strategy would give access to powerful tools to promote reactions that were not successful with either transition metal catalyst or the organocatalyst alone. The protocols presented in this thesis based on organocatalytic transformations via enamine or iminium intermediates or both, in combination with transition metal catalysis, describes new enantioselective organocatalytic procedures that afford valuable compounds with high chemo- and enantioselectivity from inexpensive commercial available starting materials. In paper I, we present a successful example of dual catalysis: the combination of transition metal activation of an electrophile and aminocatalyst activation of a nucleophile via enamine intermediate. In paper II, the opposite scenario is presented, here the transition metal activates the nucleophile and the aminocatalyst activates the electrophile via an iminium intermediate. In paper III,we present a domino Michael/carbocyclisation reaction that is catalysed by a chiral amine (via iminium/enamine activation) in combination with a transition metal catalysts activation of an electrophile. In paper IV, the concept of dual catalysis was further extended and applied for the highly enantioselective synthesis of valuable structural scaffolds, namely poly-substituted spirocyclic oxindoles. Finally, in paper V the concept of dual catalysis was expanded, by investigating more challenging and environmentally benign processes, such as the successful combination of a heterogeneous palladium and amine catalysts for the highly enantioselective synthesis of functionalised cyclopentenes, containing an all carbonquaternary stereocenter, dihydrofurans and dihydropyrrolidines.

As espécies reativas são associadas a processos toxicológicos e fisiopatológicos, agindo como importantes mediadores, por exemplo, na sinalização celular. Diversas classes de compostos têm sido utilizadas como possíveis biomarcadores de estresse redox, destacando-se os aldeídos &#945;,&#946;-insaturados, capazes de alquilar biomoléculas como o DNA. Para evitar efeitos deletérios, estes aldeídos são detoxificados por glutationilação e posterior metabolização a derivados mercaptúricos. Contudo, avaliar o estado redox em sistemas biológicos ainda é tarefa bastante complexa, sendo a dificuldade em quantificar de forma prática e acurada os efeitos de sinalização e/ou dano molecular o maior problema dos estudos redox. Assim, o objetivo deste trabalho foi desenvolver métodos acurados e sensíveis de análise de potenciais biomarcadores de estresse redox, isto é: nucleosídeos modificados, aldeídos endógenos e exógenos, glutationa e produtos de glutationilação, e avaliá-los em sistemas modelos, celular e animal, e em humanos. A avaliação dos níveis urinários de três nucleosídeos modificados por metodologia de HPLC-MS/MS desenvolvida pelo grupo em moradores da cidade de São Paulo - região com poluição atmosférica - demonstrou aumento significativo de 1,N2-propanodGuo comparado aos moradores de região não poluída. Ademais, comprova-se pela primeira vez que células deficientes em reparo de ligações cruzadas apresentam níveis basais elevados de 1,N2-propanodGuo, em duas linhagens independentes, colocando este aduto como potencial mediador de carcinogênese em pacientes portadores de Anemia de Fanconi. Utilizando cérebro de ratos SOD1G93A (modelo de Esclerose Lateral Amiotrófica - ELA), verificou-se aumento de 50% nos níveis de 1,N2-propanodGuo e de 100% nos de 1,N6-&#949;dAdo em fase sintomática, sugerindo influência do conteúdo lipídico cerebral, levando a comprometimento do metabolismo neuronal e morte celular. O perfil de aldeídos determinado em cérebro de ratos SOD1G93A demonstrou aumento de trans-hexa-2-enal e trans,trans-hexa-2,4-dienal em fase assintomática e de trans,trans-deca-2,4-dienal em fase sintomática, não sendo observada nenhuma alteração na medula. Conhecer estas variações permite direcionar estudos de modificações em biomoléculas, além de a metodologia per se corroborar com as áreas de análises lipidômicas. Técnicas distintas e o preparo de amostras refletiram nos níveis de glutationa reduzida (GSH) e oxidada (GSSG) relatados. A técnica de espectrometria de massas mostrou-se mais precisa que a detecção eletroquímica; e a alquilação do grupo tiol minimizou interferências de matriz. Por análise de HPLC-UV/Vis-ESI-MS/MS, a quantificação de trans-4-hidroxi-2-nonenal (HNE) e crotonaldeido conjugados com GSH demonstrou não haver alterações em cérebro e medula de ratos SOD1G93A. Contudo, há formação esteroespecífica dos adutos de HNE in vivo. Ressalta-se que a metodologia desenvolvida é extremamente sensível e específica e permite análise simultânea de GSH, GSSG, cisteína, cistina e dos adutos supracitados, servindo para análise de outros adutos de glutationilação de aldeídos que possam ser importantes em doenças associadas a estresse redox. / Free radicais and oxidant species are associated with toxicological and pathophysiological processes. It has been demonstrated that production of reactive oxygen species may be involved in cell signaling and regulation. Several biomarkers of redox processes have been used, including adducts formed through the reaction of &#945;,&#946;-unsaturated aldehydes with biomolecules such as DNA. In order to avoid these deleterious effects, aldehydes are detoxified through glutathionylation and further metabolized to mercapturic derivatives. However, assessing the redox status in biological systems is still a very complex task, and the difficulty in practical and accurate quantification of signaling effects and/or molecular damage is a major problem in redox studies. The objective of this work was to develop accurate and sensitive methods for analysis of potential biomarkers of redox stress, i.e., modified nucleosides, endogenous and exogenous aldehydes, glutathione and glutathionylation products, and their evaluation in cell, animal model and humans. Evaluation of urinary levels of 1,N2-propano-2\'-deoxyguanosine (1,N2-propanodGuo), 1,N2-etheno-2\'-deoxyguanosine and 8-oxo-7,8-dihydro-2\'-deoxyguanosine in residents of São Paulo City - polluted region - showed a significant increase (p<0.05) in 1,N2-propanodGuo levels compared to residents of an unpolluted region by a HPLC-MS/MS methodology developed by the group. Moreover, it was proven, for the first time, that repair deficient cells have basal levels of 1,N2-propanodGuo higher than proficient cells in two independent strains, placing 1,N2-propanodGuo as a potential mediator of carcinogenesis in Fanconi Anemia patients. In an Amyotrophic Lateral Sclerosis (ALS) animal model (SOD1G93A rat) , a 50% increase in the levels of 1,N2-propanodGuo and 100% in the 1,N6-etheno-2\'-deoxyadenosine in brain tissue in the symptomatic phase was observed, suggesting that the high brain lipid content may play a role, leading to impairment of cell metabolism and neuronal cell death. There is an increase of trans-hex-2-enal and trans,trans-hexa-2,4-dienal in asymptomatic SOD1G93A rats brain and of trans,trans-deca-2,4-dienal in symptomatic ones. However, no alteration was observed in spinal cord. Our approach contributes to a better understanding of the aldehyde status in vivo and allows us to predict biomolecule modifications. The developed methodology can contribute to lipidomic studies. The use of different techniques and sample preparation reflected in the reported levels of reduced (GSH) and oxidized glutathione (GSSG). The mass spectrometry technique proved to be more accurate than the electrochemical one, and the use of thiol alkylating agent minimizes matrix interference. No changes were observed in the levels of the GSH conjugates of trans-4-hydroxy-2-nonenal (HNE) and crotonaldehyde in brain and spinal cord of SOD1G93A rats quantified by HPLC-UV/Vis-ESI-MS/MS compared to controls. However, it was observed stereospecific HNE adducts formation in vivo. Note that this methodology is extremely sensitive and specific and allows simultaneous analysis of GSH, GSSG, Cys, cystine and the aforementioned adducts, serving for analysis of other aldehyde-glutathionylation adducts that may be important in pathologies associated with stress redox.

Danos em biomoléculas podem ocorrer a partir de uma interação direta entre as biomoléculas e espécies reativas de oxigênio e nitrogênio como também, pela reação de produtos secundários dessas espécies como eletrófilos gerados na peroxidação lipídica. Alguns desses produtos secundários possuem estabilidade química maior que as espécies reativas das quais foram derivadas e podem se ligar covalentemente as biomoléculas comprometendo o funcionamento normal das mesmas. Portanto, modificações em proteínas por aldeídos gerados na lipoperoxidação têm sido investigadas por suas implicações com desordens patológicas relacionadas à agregação proteica, e modificações em diversas vias de sinalização amplificando os efeitos deletérios em sistemas biológicos. Os objetivos desse trabalho foi contribuir na elucidação dos mecanismos moleculares associados ao desenvolvimento da esclerose lateral amiotrófica (ELA) através da identificação, caracterização e quantificação de modificações póstraducionais em proteínas pelos aldeídos 4-hidroxi-2-hexenal (HHE) e trans-4-hidroxi-2-nonenal (HNE) in vitro (citocromo c) e in vivo (modelo ELA) a partir de técnicas de Western blot, imunoprecipitação e espectrometria de massa com abordagem proteômica de \"shotgun\" em ratosSOD1G93A modelo de esclerose lateral amiotrófica (ELA). Estudos com citocromo c mostraram a ligação dos aldeídos ao citocromo c e mecanismos de reação foram propostos. Foram encontrados seis peptídeos modificados por HHE e um para o HNE, e o peptídeo TGPNLHGLFGR se mostrou modificado pelos dois aldeídos paralelamente. Foi demonstrado que a histidina 33 é um \"hot spot\" frente as adições pelos aldeídos. Nas análises por western blot das proteínas ligadas a aldeídos foi possível observar uma tendência de aumento na concentração de proteínas ligadas ao HNE nos animais ELA, mais acentuada nas amostras de 70 dias comparadas ao controle. Com relação aos resultados obtidos com HHE tanto os animais pré-sintomáticos quanto os sintomáticos não apresentaram diferenças de HHE-proteína, tantonos controles quanto nos animais ELA. Nas amostras dos animais sintomáticos não detectamos diferença significativa na concentração de aldeído-proteína entre os grupos. Já as análises proteômicas revelaram 24 proteínas diferencialmente expressas, com destaque para proteínas com os maiores valores de significância (p-value), como a ubiquitina no grupo dos pré- sintomáticos e a neurogranina, no grupo dos animais sintomáticos e várias proteínas de metabolismo energético, de neurofilamentos, proteínas de processos redox e proteínas ligadas o metabolismo de cálcio (fundamentais na fisiopatologia em ELA). Algumas proteínas importantes foram encontradas com exclusividades nos grupos pré-sintomáticos e sintomáticos pelo diagrama de Venn. Com relação a proteínas modificadas pelos aldeídos, foram encontradas algumas relevantes como a proteína 2 de interação com a polimerase delta que foi modificada por HNE via adição de Michael encontrada nos animais ELA pré-sintomáticos e sintomáticos, a catalase que foi encontrada modificada por HNE via base de Schiff apenas nos ELA pré- sintomáticos, e a tiol redutase induzível por interferon gama no grupo dos animais ELA sintomáticos. Com relação a proteínas modificadas por HHE, foram encontradas a Janus quinase e proteína 3 de interação com microtúbulo, modificadas tanto por adição de Michael quanto via base de Schiff nos animais ELA sintomáticos. É interessante ressaltar que algumas modificações encontradas em proteínas não caracterizadas podem indicar proteínas novas ainda não descritas como modificadas por esses aldeídos. Os resultados mostram que algumas das proteínas modificadas por HNE e HHE encontradas neste trabalho, estão relacionadas ao estresse redox, vias metabólicas energéticas, proteínas envolvidas na resposta a danos oxidativos, e processos inflamatórios. Tais modificações ocorrem não só no modelo de neurodegeneração, mas foram previamente descritas em outros processos patológicos, como doença cardiovascular, lesão hepática por uso crônico de álcool. / Damage to biomolecules can occur from a direct interaction between biomolecules and reactive of oxygen and nitrogen species as well as from the reaction of secondary products of these species as electrophiles generated in lipid peroxidation. Some of these by-products have greater chemical stability than the derived reactive species and can bind to biomolecules compromising their normal function. Therefore, protein modifications by aldehydes generated during lipoperoxidation have been investigated for their implications with pathological disorders related to protein aggregation and modifications in signaling pathways amplifying the deleterious effects in biological systems. The aim of this work was to contribute to the elucidation of the molecular mechanisms associated with the development of amyotrophic lateral sclerosis (ALS) through the identification, characterization and quantification of posttranslational modifications in proteins by 4-hydroxy-2-hexenal (HHE) and trans-4-hydroxy-2- nonenal (HNE) in vitro, cytochrome c, and in vivo, rat model (SOD1G93A) of amyotrophic lateral sclerosis (ALS), throught Western blot techniques, and mass spectrometry with shotgun proteomics approach. The results showed the binding of aldehydes to cytochrome c. Six peptides were modified by HHE and one by HNE. The peptide TGPNLHGLFGR was modified by the two aldehydes. Histidine 33 has been shown to be a hot spot against aldehydes additions. By western blot analysis of the aldehyde-bound proteins, it was possible to observe a tendency of increase in the concentration of HNE-bound proteins in the ALS animals, more pronounced in the samples of 70 days compared to control samples. Regarding the results obtained with HHE, both pre-symptomatic and symptomatic animals did not show HHE-protein differences, both in controls and in ALS animals. We did not detect a significant difference in the aldehyde-protein concentration between the groups in the samples of the symptomatic animals. Proteomic analysis revealed 24 differentially expressed proteins, with emphasis on proteins with thehighest values of significance (p-value), such as the ubiquitin in the pre-symptomatic group and neurogranin in the group of the symptomatic animals and several proteins of the energetic metabolism pathways, neurofilaments, proteins of redox processes and proteins linked to calcium metabolism (fundamental in the pathophysiology of ALS). Some important proteins were found exclusivity in the pre-symptomatic and symptomatic groups by the Venn diagram. With regard to aldehyde-modified proteins, some relevant ones such as Delta-2 polymerase interaction protein, that was modified by HNE via the addition of Michael found in presymptomatic and symptomatic ELA animals, catalase that was found to be modified by HNE via Schiff\'s base only in pre-symptomatic ALS, and gamma interferon-inducible thiol reductase in the group of symptomatic ALS animals. Janus kinase and microtubule interaction protein 3, were found to be modified by Michael addition and Schiff base pathway respectively in symptomatic ALS animals. It is interesting to note that some modifications found in uncharacterized proteins may indicate new proteins not yet described as modified by these aldehydes. The results show that some of the proteins modified by HNE and HHE found in this work are related to redox stress, energetic metabolic pathways, proteins involved in the response to oxidative damage, and inflammatory processes. Such modifications occur not only in the neurodegeneration model, but were previously described in other pathological processes, such as cardiovascular disease, liver injury due to chronic alcohol use

Depois dos hidrocarbonetos, os aldeídos de baixa massa molar são os mais abundantes dos gases orgânicos encontrados na atmosfera. Os aldeídos provêm de diversas fontes como as atividades industriais, incompleta combustão de combustível fóssil e biomassa e como resultado de reações fotoquímicas na atmosfera. Os aldeídos são potentes precursores de importantes oxidantes como o nitrato de peroxiacetila (PAN) e ozônio. Eles são reconhecidamente irritante dos olhos e trato respiratório, além de possuir características mutagênicas e carcinogênicas em animais. Considerando o impacto toxicológico e ambiental destes compostos, a prevenção e controle dos aldeídos requerem o uso de novas e versáteis metodologias analíticas. Neste sentido, a eletroforese capilar tem mostrado ser uma técnica alternativa para a análise de aldeídos em amostras ambientais. Este trabalho descreve diferentes metodologias desenvolvidas, em eletroforese capilar, para a separação e análise de aldeídos em amostras de ar (indoor, outdoor) e emissão veicular. As metodologias incluem a separação dos adutos aniônicos bissulfito-aldeído e das hidrazonas aniônicas formadas a partir da reação dos aldeídos com dansilhidrazina (DNSH) e ácido 4-hidrazino benzóico (HBA) por eletroforese capilar em solução livre (free solution capillary electrophoresis, FSCE), bem como a separação das hidrazonas formadas a partir da reação dos aldeídos com 2,4-dinitrofenilhidrazina (DNFH) e 3-metil-2-benzotiazolinona hidrazona (MBTH), utilizando a cromatografia eletrocinética micelar (micellar electrokinetic chromatography, MEKC) . As metodologias foram comparadas em termos de sensibilidade, limite de detecção, procedimento de amostragem, necessidade de purificação dos reagentes derivatizantes e aplicação em amostras de ar. O método do bissulfito apresentou algumas vantagens sobre os métodos estabelecidos na literatura como boa sensibilidade (limite de detecção de 3,4 - 36,9 ng mL-1), rapidez, facilidade de aplicação e pequena manipulação da amostra. A desvantagem é que requer longos tempos de amostragem para a análise de traços (ng mL-1). A metodologia com DNFH apresentou baixa sensibilidade (limite de detecção 0,14 - 2,59 &#181;g mL-1) , necessidade de purificação dos reagentes e solventes. No entanto, o sistema de pré-concentração, na coleta, tornou possível a aplicação do método a amostras de ar indoor. Usando MBTH como agente derivatizante foi possível obter limites de detecção na faixa de 3,1 - 21,1 ng mL-1, análise rápida e pouca manipulação da amostra. O reagente não requer purificação. O principal problema do método é o decréscimo do sinal analítico em função do aumento da cadeia carbônica. O método da DNSH mostrou boa sensibilidade com limites de detecção na faixa de 2,1 - 14,1 ng mL-1 para a detecção UVe 0,96 - 2,6 ng mL-1 para a detecção LIF. O reagente sofre oxidação quando as amostras não são preparadas em acetonitrila ou em outro solvente orgânico. A purificação dos solventes é necessária. O método do HBA mostrou boa sensibilidade com limites de detecção na faixa de 2,7 - 8,8 ng mL-1, rapidez e simplicidade. O solvente deve ser purificado. As hidrazonas sofrem degradação na presença de água e luz. As metodologias estudadas foram aplicadas a amostras reais de emissão veicular, ar indoor e outdoor. Foram verificadas as presenças de formaldeído, acetaldeído e acetona para amostras de ar indoor e outdoor, em concentrações na faixa de ppbv e em amostras veiculares foram encontradas formaldeído e acetaldeído em concentrações na faixa de ppmv. / After the hydrocarbons, the low molecular-weight aldehydes are the most abundant of the organic gases of the atmosphere. The aldehydes are produced from many sources such as industrial activities, incomplete combustion of fossil fuels and biomass or as result of photochemical reactions. Aldehydes are important precursor compounds of photochemical smog and their chemistry has been associated to the generation of harmful oxidants, peroxyacetylnitrate (PAN) and ozone. Aldehydes are recognized irritants of the eyes and respiratory tracts of animais and humans and often carcinogenic and mutagenic characteristics are attributed to them. Because of the toxicological and environmental importance of these compounds, prevention and control of aldehydes have demanded new and versatile analytical methodologies. In this context, capillary electrophoresis has become an interesting alternative technique for environmental analysis. This work describes different CE methodologies developed for the separation and analysis of aldehydes in environmental samples of air (indoors and outdoors) and vehicle exhaust. The methodologies comprise the free solution capillary electrophoresis separation of anionic bissulfite-aldehyde adducts, anionic aldehyde-DNSH derivatives and anionic aldehyde-HBA derivatives as well as the micellar chromatographic separation of aldehyde-DNPH derivatives and aldehyde-MBTH derivatives. These methodologies were contrasted in terms of sensitivity, Iimit of detection, sampling procedure, need for reagent purification and application to air samples. The bissulfite methodology is a novel approach with several advantages over established methods in the literature, such as good sensitivity (range from 3.4 to 36.9 ng mL-1), very easy to implement, speed of analysis and lack of sample manipulation, but it requires long collection volume of air to achieve ng mL-1 detection level. The aldehyde-DNFH derivatives methodology presented poor sensitivity (range from 0.14 to 2.59 &#181;g mL-1). The reagents and solvents must be purified to avoid contamination which will completely interfere with the sample components during analysis. However, the preconcentration achieved during sampling allowed to evaluate aldehyde levels in air samples. Using the MBTH methodology it was possible to obtain a limit of detection range from 3.1 to 21.1 ng mL-1, fast analysis and very little sample manipulation. There is not need for purification of the reagent since it is obtained in grade purity. The main problem with this reaction is that as the length of the aldehyde chain increases, the sensitivity decreases. The aldehyde-DNSH derivatives method presented good sensitivity with a limit of detection range from 2.1 to 14.1 ng mL-1 (UV detection) and 0.96 to 2.6 ng mL-1 (LIF detection). The reagent shows substantial oxidation when the sample is not prepared in acetonitrile or other organic solvent. The aldehyde-HBA derivatives method showed good sensitivity with a limit of detection range from 2.7 to 8.8 ng mL-1, it is fast and simple. However, the solvents must be purified and the derivatives shows substantial degradation in presence of water and light. The developed methodologies were then applied to real samples of indoor, outdoor and automobile emissions. The presence of formaldehyde, acetaldehyde and acetone in indoor and outdoor samples was verified at the low ppbv level and the presence of formaldehyde and acetaldehyde, in automobile samples, was at the ppmv level.

This thesis deals with the development and application of new synthetic methodology for stereo- or regioselective construction of carbon-carbon bonds in organic synthesis. The first part of this thesis describes the development of a divergent protocol for stereoselective synthesis of chiral aminodiols by employing Mukaiyama aldol additions to syn- and anti-α-amino-β-silyloxy aldehydes. The stereoselectivity of the nucleophilic attack is governed by either chelation to the α-amino moiety or by nucleophilic attack in the Felkin-Anh sense. This study is also directed towards the elucidation of the factors that dictate aldehyde π-facial selectivity in substrate-controlled nucleophilic additions to these and similar systems. In the second part, a highly stereoselective [3 + 2]-annulation reaction of N-Ts-α-amino aldehydes and 1,3-bis(silyl)propenes for stereoselective construction of densely functionalized pyrrolidines is presented. In addition, this methodology is also implemented as a keystep in a synthetic approach towards the polyhydroxylated pyrrolidine and pyrrolizidine alkaloids DGDP and (+)-alexine from a common late pyrrolidine intermediate. Finally, a divergent protocol for regioselective opening of vinyl epoxides using alkyne nucleophiles is described, in which the regioselectivity of the nucleophilic attack is controlled by the choice of reaction conditions. The regioselectivities of the SN2 and SN2’ processes are, however, significantly influenced by the nature of the alkyne substituents and the best results are obtained using ethoxyacetylene. The SN2 opening of vinyl epoxides with ethoxyacetylene as nucleophile is also shown to provide a straightforward entry to functionalized γ-butyrolactones. / QC 20100917

Chiral bisphosphinites are well-documented alternatives for chiral bisphosphines as ligands that can be exploited in various asymmpetric syntheses. Particularly, vicinal biarylphophinite ligands give a seven membered chelate ring similar to the successful DIOP on coordination to the metal. RajanBabu and coworkers have described asymmetric bisphosphinites obtained by functionalization of sugars and have shown their utility in enantioselective hydrogenation, hydrovinylation and hydrocynation reactions. Despite the interesting reactions demonstrated by bisphosphinites, not much attention has been paid to their synthesis and catalysis. This is probably due to the known moisture and oxygen sensitivity that makes their use limited. In the present thesis, a series of C1 an C2 symmetric bisphosphinite complexes of Pd(II) and Pt(II) have been synthesized directly from various naturally occurring chiral alcohols using a modified template method. A number of asymmetric catalytic reactions have been developed such as allylation of imines, allylation of aldehydes, allylic allylation, allylic alkylation, hydrosilylation of alkenes and regioselective allylation of oxiranes. Allylation of imines was carried out in essentially neutral conditions using Pd(II) catalysts and water was shown to accelerate the reaction. Interestingly acetic acid was required as a promoter in asymmetric allylation of cinnamaldehyde in the Pt(II) catalyzed reaction whereas water was a deterrent. Hydrosilylation reaction was carried out in solvent free conditions with high turnover numbers (.1000). Ascorbic acid based complexes produced the highest enantioselectivity for the asymmetric allylic alkylation reaction (97 % ee) and hydrosilylation of styrene (98% ee). These enantioselectivity results are the best obtained using ligands directly prepared from natural products.