Xanthene dyes are common fluorophores which have been widely used as molecular probes. The xanthene fluorophores can be used as highly selective optical sensors to detect disease biomarkers. A new fluorogenic dye containing an alpha, beta-unsaturated aldehyde moiety exhibits selective fluorescent signal enhancement in the presence of cysteine or peptides containing N-terminal cysteine residues. The mechanism is based on synergistic covalent and supramolecular interactions. A unique rhodamine boronic acid indicator is used in an optimized data collection protocol for wavelength- and time-dependent selectivity of various saccharides and nucleosides. One indicator is thereby capable of selectively distinguishing structurally related analytes in mixtures. Moreover, the rhodamine-based boronic acid responds linearly to increasing riboside concentrations in urine samples, potentially enabling the screening for inborn purine metabolism disorders.

Cette thèse développe la synthèse contrôlée et la purification de nanoparticules d’or hybrides, AuNPs stabilisées par des thiols organiques modulant leurs propriétés de surface. Les applications visent la catalyse et le domaine biomédical, impliquant un contrôle poussé des nanoobjets introduits. Les synthèses des AuNPs organiques sont développées à partir de la méthode de Brust, avec le 4-hydroxythiophénol et le 4-méthylthiophénol. Elles conduisent à des nanoparticules hybrides stables d’environ 2 nm. Les fractions de purifications sont analysées par MET, UV-visible, RMN et ATG, caractérisant le cœur d’or, la couche de ligands et leurs interactions. Il apparaît que les AuNPs hybrides présentent un assemblage de thiols en monocouche ou en multicouche. Une nouvelle voie de synthèse directe en phase aqueuse d’AuNPs d’environ 4 nm, stabilisées par le 4-hydroxythiophénol, est ensuite développée. Ces AuNPs sont purifiées par dialyse et caractérisées par MET, UV-visible, RMN et ATG. Les fractions d’AuNPs organiques, présentant différents états de surface, sont imprégnées dans la silice mésoporeuse SBA-15. Les isothermes d’adsorption et la manométrie sous diazote indiquent une bonne dispersion des AuNPs et une insertion dans les canaux. Nous introduisons l’exploration d’applications ciblées. L’utilisation des AuNPs organiques lors de l'oxydation d’alcènes tend à améliorer la sélectivité du sel de manganèse catalytique. Pour le domaine biomédical, les AuNPs aqueuses présentent une bonne dispersibilité en milieux aqueux biocompatibles. Les premiers tests in-vitro sur des cellules de sarcomes humains montrent une faible cytotoxicité et une bonne pénétration intracellulaire. / This Ph.D. work developed the controlled synthesis and purification of hybrid gold nanoparticles AuNPs, stabilized by organic thiols that are tuning their surface properties. The targeted applications are the catalysis and in the biomedical field, requiring a thorough control of the introduced nanoobjects. Syntheses of the organic AuNPs were developed from the Brust method, using 4-hydroxymercaptophenol or 4-methylmercaptophenol, leading to stable hybrid gold nanoparticles of size 2 nm. Purified fractions were characterized using TEM, UV-visible, NMR and TG analysis, issuing key data about the gold core, the organic layer and their interactions. Among the different fractions of AuNPs, the organic thiol ligands appeared to be assembled either as a monolayer or a multilayer pattern. A new direct route for synthesis of aqueous AuNPs of size 4 nm, stabilized by 4-hydroxymercaptophenol, has been developed. The AuNPs were purified using dialysis and characterized by TEM, UV-visible, NMR and TG analysis. Organic AuNPs, exhibiting different surface properties, were impregnated into SBA-15 mesoporous silica. Adsorption isotherms and nitrogen adsorption/desorption studies were in good agreement with the homogeneous distribution of AuNPs and the significant incorporation into the porosity. Finally, exploration of the targeted applications was started. The use of organic AuNPs for alkene oxidation tends to improve the selectivity of manganese salt catalyst. In the biomedical field, the aqueous AuNPs exhibited good dispersibility into biocompatible aqueous solvents. First in-vitro assays involving human sarcoma cells line showed limited cytotoxicity and good cellular uptake.

Les macrocycles peptidiques sont des molécules ayant une importance pharmaceutique grandissante. Le cycle restreint le nombre de conformations accessibles et peut potentiellement améliorer un certain nombre de paramètres pharmacocinétiques et pharmacodynamiques, en comparaison avec un analogue linéaire. La synthèse de macrocycles exige de favoriser la réaction intramoléculaire par rapport à la réaction intermoléculaire, ce qui est souvent réalisé par dilution du milieu dans l’ordre du mM. Les contraintes de concentration rendent certaines réactions de macrocyclisation peu pratiques. Par exemple, les processus photochimiques sont intrinsèquement sensibles à la concentration et à la pénétration de la lumière. Leur efficacité peut être améliorée en réduisant la longueur du trajet optique de la lumière dans une solution, mais cette solution est difficilement transposable aux procédés de macrocyclisation. Le chapitre 1 de ce mémoire introduit les concepts de macrocyclisation et de réaction photochimique. Le chapitre 2 introduit, d’une part, les réactions de macrocyclisation photochimique et, d’une autre, le travail de conception réalisé par Émilie Morin d’un réacteur hybride en flux continu favorisant ce type de réaction. Le chapitre 3 explicite les démarches effectuées pour réaliser les objectifs de ce mémoire. Plus précisément, l’oxydation aérobique photocatalytique de thiols en disulfure dans le réacteur hybride permet l’isolation de cinq macrocycles avec de meilleurs rendements qu’en montage en batch. Le couplage de Glaser-Hay est utilisé pour dériver un des macrocycles synthétisés dans le réacteur hybride. Enfin, l’investigation préliminaire de deux autres systèmes photocatalytique/photochimique est abordée pour leur application future dans le réacteur nouvellement développé. / Peptide macrocycles are molecules of increasing pharmaceutical importance. Their restricted conformations can potentially improve a number of pharmacokinetic and pharmacodynamic parameters, compared to their linear analogs. The synthesis of macrocycles requires favoring the intramolecular reaction over the intermolecular reaction, which is often achieved by dilution in the order of mM. The constraints of concentration make certain macrocyclization reactions unpractical. For example, photochemical processes are inherently sensitive to concentration and light penetration. Their efficiency can be improved by reducing the length of the optical path of the light in a solution, but the solution is difficult to transfer to macrocyclization processes. Chapter 1 of the thesis introduces the concepts of macrocyclization and photochemical reactions. Chapter 2 introduces photochemical macrocyclization reactions and the design work carried out by Émilie Morin of a continuous flow hybrid reactor. Chapter 3 explains the steps taken to achieve the objectives of the thesis. More specifically, the photocatalytic aerobic oxidation of thiols to disulfide in the hybrid reactor allows the isolation of five macrocycles with better yields than in batch. The Glaser-Hay coupling is used to derivatize one of the macrocycles synthesized in the hybrid reactor. Finally, the preliminary investigation of two other photocatalytic/photochemical systems is discussed for their future application in the newly developed reactor.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2011. / Includes bibliogaphy. / ENGLISH ABSTRACT: Sauvignon blanc wines have become increasingly popular in South Africa as it is a cultivar that can be easily manipulated in the vineyard and cellar to produce a range of wine styles. These wines are usually given aroma descriptors such as green pepper, grassy and asparagus; while other more tropical aromas include passion fruit and guava. These aromas are thought to be mainly caused by methoxypyrazines and volatile thiols. These compounds are known to be character impacting compounds of Sauvignon blanc and are present in the grapes in the aromatic form (methoxypyrazines) or as non‐aromatic precursors (thiols) that can be released by the yeast during fermentation. Other aroma compounds such as esters, higher alcohols, fatty acids and monoterpenes are compounds that could potentially influence the aroma bouquet of a wine significantly. These aroma compounds exist either as precursors in the grapes (monoterpenes) or arise due to yeast metabolism during fermentation (esters, higher alcohols, fatty acids) and often display fruity, floral and pleasant aromas. In the cellar, winemaking practices can be manipulated to a certain extent to achieve the desired wine style. Winemaking tools such as temperature, skin contact, pressing conditions, oxygen (O2), sulphur dioxide (SO2) and yeast strain are only a few factors influencing the outcome of a wine. In general, South African winemakers maintain a very reductive environment during Sauvignon blanc wine production by using inert gasses, thereby causing the production costs to increase. There is sufficient evidence to support the reductive handling of white wine, however there seems to be a lack of information as to why the must should be treated reductively before fermentation. The over all goal of this study was thus to investigate the effect of different O2 and SO2 additions to Sauvignon blanc must before settling, specifically focussing on the typical aroma compounds often found in these wines. Chapter 2 gives an overview of the oxidation reactions occurring in must (enzymatic oxidation) and wine (chemical oxidation). This chapter also reports the origin of the specific Sauvignon blanc aroma compounds and their reaction to different must and wine treatments with a focus on oxidation. Chapter 3 reports research results focussing on the effect of the different must treatments on the character impacting compounds of Sauvignon blanc wines, specifically the methoxypyrazines and the volatile thiols. The effect of the treatments on the polyphenols and glutathione content in the must and wine was also investigated. Oxidation in the absence of SO2 led to a decrease in glutathione and certain phenolic compounds in the must. In general, volatile thiols were protected against oxidation by SO2, even when O2 was present in the must. Methoxypyrazines concentrations were not significantly influenced by the treatments. Chapter 4 elucidates the effect of the treatments on other yeast and grape derived aroma compounds often found in Sauvignon blanc wines, such as the esters, higher alcohols, fatty acids and monoterpenes. In general, the effect of SO2 seemed to have the greatest influence on the produced aroma compounds. The results reported in this thesis could possibly change the way South African Sauvignon blanc musts are handled in future during the winemaking process. It is clear that O2 and SO2 management in the cellar is of critical importance for the winemaker to produce wines of high quality. Future work is important to fully understand the mechanisms and evolution of important aroma compounds of Sauvignon blanc wines during the winemaking process. / AFRIKAANSE OPSOMMING: Sauvignon blanc wyn aroma word gewoonlik beskryf met terme soos groen rissie, grasagtig en aspersie terwyl ander tropiese aromas soos grenadella en koejawel ook dikwels voorkom. Die manipulasie van Sauvignon blanc in die wingerd en in die kelder tydens wynmaak, gee die wynprodusent die vryheid om ‘n wye reeks wyn style te produseer. Dit maak Sauvignon blanc baie populêr in die Suid‐Afrikaanse wynindustrie. Die bogenoemde aromas word waargeneem in die wyn as gevolg van die teenwoordigheid van sekere aroma komponente genaamd metoksipirasiene en vlugtige tiole. Hierdie komponente lewer ‘n unieke bydrae tot die aroma samestelling van Sauvignon blanc wyne en kom voor in die druiwe in die aromatiese vorm (metoksipirasiene) of as nie‐aromatiese voorlopers (tiole) wat tydens alkoholiese fermentasie deur die gis vrygestel kan word. Komponente soos esters, hoër alkohole, vetsure en monoterpene kan ook ‘n potensiële bydra lewer tot die algehele aroma van Sauvignon blanc wyne en kom voor in die druiwe (monoterpene) of ontstaan as gevolg van gis metabolisme gedurende alkoholiese fermentasie (esters, hoër alkohole, vetsure). Hierdie geur komponente word dikwels beskryf as vrugtig, blomagtig en oor die algemeen aangenaam. Tydens wynmaak kan die wyn tot ‘n mate gemanipuleer word om ‘n spesifieke wynstyl te bekom. Hulpmiddels soos temperatuur, dopkontak, pers omstandighede, suurstof (O2), swawel dioksied (SO2) en gisras is slegs ‘n paar faktore wat die algemene uitkoms van ‘n wyn kan beïnvloed. Oor die algemeen word Sauvignon blanc in Suid‐Afrika baie reduktief behandel tydens wynbereiding. Dit vereis sekere hulpmiddels, soos die gebruik van inerte gas, wat die produksiekoste dikwels verhoog. Navorsing ondersteun die reduktiewe behandeling van wit wyn, maar dit wil voorkom asof daar ‘n tekort aan navorsing is wat die reduktiewe behandeling van die sap voor fermentasie regverdig. Die algemene doel van die studie is dus om die effek van verskillende O2 en SO2 byvoegings tot Sauvignon blanc sap (voor afsak) te ondersoek met die fokus op die tipiese aroma komponente wat in die wyn voorkom. Hoofstuk 2 lewer ‘n algemene oorsig van die tipes oksidasie reaksies wat voorkom in sap (ensiematiese oksidasie) en wyn (chemiese oksidasie). Spesifieke Sauvignon blanc aroma komponente word ook ondersoek in terme van die oorsprong van die komponente asook die reaksie wat plaasvind met verskillende mos en wyn behandelings, met ‘n fokus op oksidasie. In hoofstuk 3 word die effek van die verskillende mos behandelings op tipiese Sauvignon blanc aroma komponente, spesifiek metoksipirasiene en vlugtige tiole, ondersoek. Die effek van die behandelings op die polifenole en glutatioon inhoud in die mos en wyn word ook gerapporteer. Oksidasie van die sap in die afwesigheid van SO2, het ‘n afname in glutatioon en sekere polifenol konsentrasies veroorsaak. Dit wil voorkom asof die produksie van vlugtige tiole oor die algemeen beskerm word teen oksidasie indien daar genoegsame SO2 teenwoordig is. Hierdie effek word ondervind selfs as die sap met suursof versadig word. Die effek van die behandelings op die konsentrasies van metoksipirasiene was nie beduidend nie. Hoofstuk 4 rapporteer die effek van die behandelings op ander aroma komponente soos esters, hoër alkohole, vetsure en monoterpene. Oor die algemeen wil dit voorkom asof die effek van SO2 beduidend was en waarskynlik die grootste invloed op die produksie van hierdie aroma komponente het. Na aanleiding van die resultate bevind in hierdie tesis, is daar ‘n moontlikheid dat die manier waarop Sauvignon blanc wyne geproduseer word in Suid‐Afrika, moontlik kan verander in die toekoms. Vir die wynmaker om hoë kwaliteit Sauvignon blanc wyne te produseer, is O2 en SO2 bestuur in die kelder van kardinale belang. Verdere navorsing moet steeds gedoen word om die meganisme en evolusie van belangrike aroma komponente in Sauvignon blanc wyne tydens die wynmaakproses, ten volle te verstaan.

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Sauvignon blanc wines have become progressively more important in the commercial market. Extensive research is being done in various countries to gain more understanding about the aroma compounds found in Sauvignon blanc wines and the interactions between them. Sauvignon blanc wines often have either have a green or tropical style. The green style is caused by the methoxypyrazines while the volatile thiols are important contributing compounds to the tropical style. Various international studies have focussed on measuring the chemical composition of Sauvignon blanc wines. However, more research is required on South African Sauvignon blanc wines. Little is known of the volatile thiols content of South African Sauvignon blanc wines, although the methoxypyrazine content has been extensively reported on. Although methoxypyrazines and volatile thiols are seen as the most important aroma compounds contributing to Sauvignon blanc character, other compounds contribute as well. Esters, monoterpenes and phenols have been found to influence Sauvignon blanc aroma and interact with the methoxypyrazines and volatile thiols. The complex interaction between the compounds responsible for the aroma of Sauvignon blanc wines are still not fully understood and further research is thus needed. The first part of the current study investigated the interaction between a specific methoxypyrazine and volatile thiol. Five different concentrations of 2-isobutyl-3- methoxypyrazine (ibMP) and 3-mercaptohexan-1-ol (3MH) were spiked in dearomatize, neutral Sauvignon blanc wine. The single compounds as well as every possible combination of the range of concentrations were evaluated using sensory descriptive analysis. It was found using various statistical approaches that ibMP suppressed the tropical attributes associated with 3MH and that 3MH suppressed the green attributes that correlated with ibMP. The concentrations at which the suppression occurred and the degree of suppression was different for each attribute. The second part of the current study focussed on commercial South African Sauvignon blanc wines. Sensory descriptive analysis and chemical analysis were used to assess the wines and measure the volatile thiol and methoxypyrazine concentrations. The concentrations of volatile thiols and methoxypyrazines were found to be in line with international Sauvignon blanc wines. It was also shown for the first time that the mutually suppressive trend between the volatile thiols and methoxypyrazines can be seen in commercial Sauvignon blanc wines as well. Future work is needed to fully understand the complex interaction between the various compounds in Sauvignon blanc wines. Further research could focus on investigating the mechanism of interaction between the volatile thiols and methoxypyrazines as well as other aroma compounds. / AFRIKAANSE OPSOMMING: Sauvignon blanc-wyne word toenemend belangriker in die kommersiële mark. Omvattende navorsing word in etlike lande gedoen om meer begrip te ontwikkel van die aromaverbindings wat in Sauvignon blanc-wyne teenwoordig is, asook van die interaksies tussen hulle. Sauvignon blanc-wyne het in baie gevalle óf ’n groen óf ’n tropiese styl. Die groen styl word veroorsaak deur metoksipirasiene, terwyl die vlugtige tiole belangrike bydraende verbindings is wat aanleiding gee tot die tropiese style. Verskeie internasionale studies het reeds gefokus op die meet van die chemiese samestelling van Sauvignon blanc-wyne, maar meer navorsing is nodig oor Suid-Afrikaanse Sauvignon blanc-wyne. Min is bekend oor die inhoud van vlugtige tiole in Suid-Afrikaanse Sauvignon blanc-wyne, hoewel daar reeds op groot skaal oor die metoksipirasieninhoud verslag gedoen is. Hoewel metoksipirasiene en vlugtige tiole die belangrikste aromaverbindings is wat tot Sauvignon blanc-karakter bydra, is daar ook ander verbindings wat ’n bydrae maak. Esters, monoterpene en fenole het almal ’n invloed op Sauvignon blanc-aroma en reageer op die metoksipirasiene en vlugtige tiole. Die komplekse interaksie tussen die verbindings wat vir die aroma van Sauvignon blanc-wyne verantwoordelik is, word nog nie volledig begryp nie en verdere navorsing is nodig. Die eerste deel van die huidige studie het die interaksie tussen ’n spesifieke metoksipirasien en vlugtige tiol ondersoek. Vyf verskillende konsentrasies van 2-isobutiel-3-metoksipirasien (ibMP) en 3-merkaptoheksaan- 1-ool (3MH) is by ontgeurde, neutrale Sauvignon blanc-wyn gevoeg. Die enkel verbindings, asook elke moontlike kombinasie van die reeks konsentrasies, is deur middel van beskrywende sensoriese analise geëvalueer. Daar is met behulp van verskillende statistiese benaderings gevind dat ibMP die tropiese eienskappe wat verband hou met 3MH onderdruk het, terwyl 3MH die groen eienskappe wat verband hou met ibMP onderdruk het. Die konsentrasies waarteen die onderdrukking plaasgevind het en die vlak van onderdrukking het vir elke eienskap verskil. Die tweede deel van die studie het gefokus op kommersiële Suid-Afrikaanse Sauvignon blancwyne. Beskrywende sensoriese analise en chemiese analise is gebruik om die wyne te assesseer en die konsentrasies van vlugtige tiole en metoksipirasiene te meet. Die konsentrasies vlugtige tiole en metoksipirasiene was in lyn met dié van internasionale Sauvignon blanc-wyne. Daar is ook vir die eerste keer gewys dat die wedersyds onderdrukkende tendens tussen die vlugtige tiole en metoksipirasiene ook in kommersiële Sauvignon blanc-wyne gevind word. Toekomstige werk sou kon fokus op ’n begrip van die komplekse interaksie tussen die verskillende verbindings in Sauvignon blanc-wyne. Verdere navorsing sou ook kon fokus op ’n ondersoek van die meganisme van interaksie tussen die vlugtige tiole en metoksipirasiene, sowel as ander aromaverbindings. / The South African Wine Industry (Winetech), THRIP and the NRF for financial support

Les thiols à faible masse moléculaire (FMM) peuvent affecter la biodisponibilité du mercure et mener à une plus grande production de méthylmercure par les microorganismes méthylants. Les résultats d’une étude réalisée au sein de la matrice extracellulaire du périphyton de la zone littorale d’un lac des Laurentides ont démontré que les concentrations en thiols à FMM retrouvées dans cette matrice sont jusqu’à 1000 fois supérieures à celles de la colonne d’eau avoisinante. Ces thiols sont significativement corrélés à la chlorophylle a du périphyton, suggérant une production par les algues. Le mercure de la matrice extracellulaire, plus spécifiquement dans la fraction colloïdale mobile, est aussi corrélé aux thiols d’origine algale. Ces résultats suggèrent qu’une accumulation de thiols s’opère dans l’espace extracellulaire du périphyton et qu’ils peuvent favoriser le transfert du mercure vers les microorganismes produisant le méthylmercure. Les résultats d’une seconde étude menée sur les méthodes de préservation d’échantillons d’eau pour la conservation des thiols à FMM ont démontré que la température optimale d’entreposage était de -80 °C et que la lyophilisation menait à une sous-estimation des concentrations mesurées. Les taux de dégradation étaient plus rapides lors de la conservation à -20 °C et variables selon la chimie de l’eau utilisée et les espèces de thiols observées. Suivant ces résultats, nous proposons un cadre de recherche pour de futures études sur la spéciation du mercure dans le périphyton et nous suggérons des précautions lors de la manipulation et de la conservation des thiols à FMM d’échantillons naturels. / Low molecular weight (LMW) thiols can affect the bioavailability of mercury and by enhancing methylmercury production by methylating microorganisms. Results of a study conducted within periphyton extracellular matrix in the littoral zone of a lake of the Laurentians have shown that LMW thiol concentrations found in this matrix are up to 1000 fold higher than those in the water column of surrounding waters. Relationships between these thiols and periphyton chlorophyll a were significant, suggesting production by algae. Mercury in the extracellular matrix, especially in the mobile colloidal fraction, was also correlated to thiols from algal sources. These results suggest thiol accumulation in the periphyton extracellular space, and that thiols likely promote mercury transfer to methylating microorganisms. A second study on preservation methods of natural water samples for LMW thiols conservation showed that a storage temperature of -80 °C was optimal and that freeze-drying led to an under-estimation of measured concentrations. Degradation rates were faster with storage at -20 °C and varied substantially depending on the chemistry of the water used as depending on thiol species observed. In regards of these results, we propose a research framework for future studies on mercury speciation processes in periphyton. We also suggest precautions on handling and storing natural samples for LMW thiol analyses.

Os lipídeos abrangem uma ampla gama de moléculas hidrofóbicas presentes nas células. As características moleculares dos lipídios determinam sua localização celular e função biológica. Em geral, os lipídios são considerados componentes essenciais de membranas, reservatórios de energia e moduladores de vias de sinalização ligadas ao metabolismo celular, sobrevivência, entre outros. Em mamíferos, grande parte dos lipídios é esterificada em ácidos graxos poli-insaturados (PUFAs), especialmente os ácidos docosahexaenóico (DHA) e araquidônico (ARA), essenciais para vários processos fisiológicos, incluindo o desenvolvimento normal do cérebro. No entanto, os PUFAs são muito suscetíveis à oxidação por espécies reativas de oxigênio (ROS) geradas endogenamente. Uma vez oxidados, lipídios são capazes de modificar grupos tióis de peptídeos e proteínas, levando à modulação das vias de sinalização e alterando o balanço redox celular. No capítulo 1, foram investigados os mecanismos envolvidos na modificação de grupos tióis de peptídeos e proteínas por produtos de auto-oxidação de PUFAs. Com as análises realizadas foi possível identificar vários adutos de glutationa (GSH) covalentemente modificados por endoperóxidos cíclicos derivados de DHA e ARA. Uma análise detalhada dos espectros de MS/MS dos adutos de GSH revelou que GSH e endoperóxidos cíclicos são provavelmente ligados através de uma ligação química de enxofre-oxigênio, em uma reação que envolve um ataque nucleofílico do ânion tiolato. Além disso, sugerimos que a eficiência da modificação do tiol por endoperóxidos cíclicos também é dependente da reatividade do tiol, como demonstrado pela modificação covalente do resíduo de cisteína mais reativo (Cys111) da enzima antioxidante superóxido dismutase 1(SOD1). Modificações químicas de tióis por endoperóxidos cíclicos podem modular a agregação proteica e o status redox celular, produzindo adutos de GSH capazes de modular a inflamação, como relatado para os conjugados de GSH gerados enzimaticamente. No capítulo 2, nós investigamos o papel dos lipídios na esclerose lateral amiotrófica (ALS), uma vez que a inflamação e o estresse oxidativo nos neurônios motores contribuem para o desenvolvimento desta doença neurodegenerativa. Usando uma abordagem lipidômica não direcionada baseada em espectrometria de massa acoplada à cromatografia líquida (UHPLC-MS/MS), nós investigamos o metabolismo lipídico no córtex motor e na medula espinhal de um modelo de ratos com ALS. A análise do córtex motor mostrou que as principais alterações lipídicas foram dependentes da idade e ligadas ao metabolismo dos esfingolipídios. Em contraste, as principais alterações lipídicas na medula espinhal foram encontradas no grupo sintomático da ALS, sendo o metabolismo de ceramidas, ésteres de colesterol e cardiolipinas os mais afetados. De acordo com os resultados obtidos e dados relatados na literatura, propusemos um mecanismo baseado em neuroproteção que envolve o acúmulo de ésteres de colesterol esterificados em PUFAs em astrócitos. Coletivamente, nossos achados sugerem que os lipídios desempenham um papel crucial na modulação de processos celulares ligado à oxidação de tióis e à neurodegeneração. / Lipids encompass a wide range of hydrophobic molecules present in cells. The molecular characteristics of lipids determine their cellular localization and biological function. In general, lipids are regarded as essential components of membranes, as energy reservoir and modulators of signaling pathways linked to cellular metabolism and survival, among others. In mammals, a large part of the lipids are esterified to polyunsaturated fatty acids (PUFAs), especially docosahexaenoic (DHA) and arachidonic (ARA) acids, essential for several physiological processes, including normal brain development. However, PUFAs are very susceptible to oxidation by reactive oxygen species (ROS) generated endogenously. Once oxidized, lipids are able to modify thiol groups of peptides and proteins leading to modulation of signaling pathways and cellular redox balance. In the chapter 1, we investigated the mechanisms involved in modification of thiol groups of peptides and protein by autoxidation products derived from PUFAs. Here, we identified several glutathione (GSH) adducts covalently modified by hydroxy-endoperoxides derived from both DHA and ARA. Detailed inspection of MS/MS spectra of GSH-adducts revealed that GSH and hydroxy-endoperoxides are likely bonded through a sulfur-oxygen chemical bond in a reaction which involves a nucleophilic attack by the thiolate anion. Also, we suggest that the efficiency of modification of thiol by hydroxy-endoperoxides are also dependent of the thiol reactivity, as demonstrated by covalent modification of the most reactive cysteine residue (Cys111) of the antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1). Chemical modifications of thiol groups by hydroxy-endoperoxides may modulate protein aggregation and cellular redox status, yieldingGSH adducts capable to modulate inflammation, as reported for the enzymatically generated counterparts. In the chapter 2, we investigated the role of lipids in amyotrophic lateral sclerosis (ALS), since inflammation and oxidative stress in motor neurons are hallmarks of this neurodegenerative disease. Using an untargeted lipidomics approach based on mass spectrometry coupled to liquid chromatography (UHPLC-MS/MS), we investigated the lipid metabolism in motor cortex and spinal cord tissues of a rodent model of ALS. Analysis of the motor cortex showed that the main lipid alterations were age-dependent and linked to metabolism of sphingolipids. In contrast, the major lipid alterations in the spinal cord were found in ALS symptomatic group, being the metabolism of ceramides, cholesteryl esters and cardiolipin the most affected. According to our findings and data reported in the literature, we proposed a mechanism based on neuroprotection that involves accumulation of cholesteryl esters esterified to PUFAs in astrocytes. Collectively, our findings suggest that lipids play a crucial role in modulation of cellular process linked to thiol metabolism and neurodegeneration.

Les transporteurs ABC forment une vaste famille de protéines présentes dans tous les organismes vivants. Ces protéines utilisent l'énergie fournie par l'hydrolyse de l'ATP pour transporter à travers les membranes biologiques des substances très variées. Plusieurs protéines ABC sont importantes pour la santé humaine. Par exemple, le défaut fonctionnel de la protéine CFTR cause la mucoviscidose et la surproduction de protéine MRP1 est associée aux phénomènes de résistance aux traitements anti-tumoraux. La levure Saccharomyces cerevisiae possède une famille de protéines (Yor1p, Ycf1p, Bpt1p, Ybt1p, Vmr1p, Nft1p) apparentées à CFTR et MRP1. Cette famille peut servir de modèle à l'étude des protéines humaines. La première partie de ce travail de thèse a été consacrée à l'étude biochimique de la protéine de levure Yor1p. Nous avons fusionné YOR1 avec un fragment d'ADN codant un peptide de poly-histidine et avons placé cette construction sous contrôle d'un promoteur permettant une surproduction dans la levure. Nous avons alors montré que la protéine Yor1p poly-histidylée était produite sous forme fonctionnelle dans la levure, puis avons mis au point une méthode permettant de solubiliser puis de purifier cette protéine en une seule étape par chromatographie d'affinité sur une colonne greffée avec des ions métalliques. La deuxième partie de ce travail a consisté à produire sous forme isolée chez la bactérie Escherichia coli et à purifier à homogénéité les deux domaines de Yor1p impliqués dans la liaison et l'hydrolyse de l'ATP. Nous avons étudié la fixation de l'ATP sur ces deux domaines, ce qui nous a permis de conclure que ces domaines étaient bien structurés. Ils peuvent maintenant être utilisés pour des études structurales. Enfin, nous nous sommes intéressés au rôle la protéine Ycf1p dans la détoxication du sélénite. Nous avons observé que la toxicité du sélénite pour la levure était considérablement accrue par la présence de composés thiolés dans le milieu de culture. La formation de dérivés réactifs de l'oxygène est vraisemblablement à l'origine de cette hypertoxicité.

Os lipídeos abrangem uma ampla gama de moléculas hidrofóbicas presentes nas células. As características moleculares dos lipídios determinam sua localização celular e função biológica. Em geral, os lipídios são considerados componentes essenciais de membranas, reservatórios de energia e moduladores de vias de sinalização ligadas ao metabolismo celular, sobrevivência, entre outros. Em mamíferos, grande parte dos lipídios é esterificada em ácidos graxos poli-insaturados (PUFAs), especialmente os ácidos docosahexaenóico (DHA) e araquidônico (ARA), essenciais para vários processos fisiológicos, incluindo o desenvolvimento normal do cérebro. No entanto, os PUFAs são muito suscetíveis à oxidação por espécies reativas de oxigênio (ROS) geradas endogenamente. Uma vez oxidados, lipídios são capazes de modificar grupos tióis de peptídeos e proteínas, levando à modulação das vias de sinalização e alterando o balanço redox celular. No capítulo 1, foram investigados os mecanismos envolvidos na modificação de grupos tióis de peptídeos e proteínas por produtos de auto-oxidação de PUFAs. Com as análises realizadas foi possível identificar vários adutos de glutationa (GSH) covalentemente modificados por endoperóxidos cíclicos derivados de DHA e ARA. Uma análise detalhada dos espectros de MS/MS dos adutos de GSH revelou que GSH e endoperóxidos cíclicos são provavelmente ligados através de uma ligação química de enxofre-oxigênio, em uma reação que envolve um ataque nucleofílico do ânion tiolato. Além disso, sugerimos que a eficiência da modificação do tiol por endoperóxidos cíclicos também é dependente da reatividade do tiol, como demonstrado pela modificação covalente do resíduo de cisteína mais reativo (Cys111) da enzima antioxidante superóxido dismutase 1(SOD1). Modificações químicas de tióis por endoperóxidos cíclicos podem modular a agregação proteica e o status redox celular, produzindo adutos de GSH capazes de modular a inflamação, como relatado para os conjugados de GSH gerados enzimaticamente. No capítulo 2, nós investigamos o papel dos lipídios na esclerose lateral amiotrófica (ALS), uma vez que a inflamação e o estresse oxidativo nos neurônios motores contribuem para o desenvolvimento desta doença neurodegenerativa. Usando uma abordagem lipidômica não direcionada baseada em espectrometria de massa acoplada à cromatografia líquida (UHPLC-MS/MS), nós investigamos o metabolismo lipídico no córtex motor e na medula espinhal de um modelo de ratos com ALS. A análise do córtex motor mostrou que as principais alterações lipídicas foram dependentes da idade e ligadas ao metabolismo dos esfingolipídios. Em contraste, as principais alterações lipídicas na medula espinhal foram encontradas no grupo sintomático da ALS, sendo o metabolismo de ceramidas, ésteres de colesterol e cardiolipinas os mais afetados. De acordo com os resultados obtidos e dados relatados na literatura, propusemos um mecanismo baseado em neuroproteção que envolve o acúmulo de ésteres de colesterol esterificados em PUFAs em astrócitos. Coletivamente, nossos achados sugerem que os lipídios desempenham um papel crucial na modulação de processos celulares ligado à oxidação de tióis e à neurodegeneração. / Lipids encompass a wide range of hydrophobic molecules present in cells. The molecular characteristics of lipids determine their cellular localization and biological function. In general, lipids are regarded as essential components of membranes, as energy reservoir and modulators of signaling pathways linked to cellular metabolism and survival, among others. In mammals, a large part of the lipids are esterified to polyunsaturated fatty acids (PUFAs), especially docosahexaenoic (DHA) and arachidonic (ARA) acids, essential for several physiological processes, including normal brain development. However, PUFAs are very susceptible to oxidation by reactive oxygen species (ROS) generated endogenously. Once oxidized, lipids are able to modify thiol groups of peptides and proteins leading to modulation of signaling pathways and cellular redox balance. In the chapter 1, we investigated the mechanisms involved in modification of thiol groups of peptides and protein by autoxidation products derived from PUFAs. Here, we identified several glutathione (GSH) adducts covalently modified by hydroxy-endoperoxides derived from both DHA and ARA. Detailed inspection of MS/MS spectra of GSH-adducts revealed that GSH and hydroxy-endoperoxides are likely bonded through a sulfur-oxygen chemical bond in a reaction which involves a nucleophilic attack by the thiolate anion. Also, we suggest that the efficiency of modification of thiol by hydroxy-endoperoxides are also dependent of the thiol reactivity, as demonstrated by covalent modification of the most reactive cysteine residue (Cys111) of the antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1). Chemical modifications of thiol groups by hydroxy-endoperoxides may modulate protein aggregation and cellular redox status, yieldingGSH adducts capable to modulate inflammation, as reported for the enzymatically generated counterparts. In the chapter 2, we investigated the role of lipids in amyotrophic lateral sclerosis (ALS), since inflammation and oxidative stress in motor neurons are hallmarks of this neurodegenerative disease. Using an untargeted lipidomics approach based on mass spectrometry coupled to liquid chromatography (UHPLC-MS/MS), we investigated the lipid metabolism in motor cortex and spinal cord tissues of a rodent model of ALS. Analysis of the motor cortex showed that the main lipid alterations were age-dependent and linked to metabolism of sphingolipids. In contrast, the major lipid alterations in the spinal cord were found in ALS symptomatic group, being the metabolism of ceramides, cholesteryl esters and cardiolipin the most affected. According to our findings and data reported in the literature, we proposed a mechanism based on neuroprotection that involves accumulation of cholesteryl esters esterified to PUFAs in astrocytes. Collectively, our findings suggest that lipids play a crucial role in modulation of cellular process linked to thiol metabolism and neurodegeneration.

Les matériaux agroressourcés à base de gluten de blé présentent des propriétés mécaniques qui ne leurs permettent pas de concurrencer celles des plastiques usuels issus de la pétrochimie. Les objectifs de ce travail de thèse visent (i) à améliorer les propriétés d'élongation et de résistance des matériaux gluten pour atteindre celles des polymères courants et (ii) à maîtriser la réactivité du gluten au cours de l'élaboration des matériaux afin de pouvoir utiliser les procédés connus de la plasturgie. L'enjeu scientifique est de comprendre la réactivité du gluten sous l'effet des traitements thermomécaniques et les mécanismes régissant les propriétés mécaniques des matériaux. Les fonctions réactives visées sont les thiols/disulfures qui assurent la réticulation des protéines du gluten. Nous avons testé l'effet de bloqueurs de thiols de type maléimide mono- et bifonctionnels, de nature, de taille et d'hydrophobicité variées. Ces derniers ont éventuellement été bloqués par réaction de Diels-Alder. L'ajout d'additifs de type bismaléimide bloqué par réaction de Diels-Alder permet de différer la réticulation à l'étape de thermoformage et de substituer aux liens covalents habituels des liens thioéthers. L'ajout de cet additif permet de doubler l'élongation à la rupture du matériau gluten mais entraîne la chute de la rigidité. L'effet de l'ajout de molécules bis- et tétrathiols a également été testé. Ces additifs ont permis d'augmenter par plus de 1,5 fois l'élasticité des matériaux gluten. Une analyse multi-échelle (moléculaire par FTIR, macromoléculaire par SEC et macroscopique par test de traction ; le tout complété par une analyse DMTA) de la structure et des propriétés a montré que l'absence de gain en élasticité était due au maintien d'une organisation structurale majoritaire en hélices-α, qui est le propre du gluten natif. La création d'interactions interprotéiques par feuillets-β a été identifiée comme seule responsable du gain d'élasticité des matériaux, la formation d'agrégats protéiques par le biais de liaisons disulfures ou thioéthers ne jouant qu'un rôle secondaire. Un mécanisme réactionnel mettant en avant les conditions qui assurent la participation de toutes les classes de protéines du gluten à la constitution du réseau protéique est discuté. Deux nouvelles voies prometteuses de mélange avec du caoutchouc et copolymérisation par « grafting from » ont été explorées et restent à approfondir. / Wheat gluten can be used to make biomaterials. Nevertheless their mechanical properties are not competitive with commonly used petroleum-based plastics. The purposes of this work aim at (i) improving strain and strength properties of gluten-based materials in order to reach those of common polymers and (ii) controlling gluten reactivity during material process in order to use already well-known processes for manufacturing plastics. The scientific stakes are to understand gluten reactivity during thermo-mechanical treatments and the mechanisms which govern mechanical properties of materials. The reactive functions of gluten are thiols/disulfides which are responsible of gluten proteins crosslinking. The effect of thiol blocker molecules such as mono and bismaleimide of various nature, sizes and hydrophobicity was tested. These molecules were eventually blocked by Diels-Alder reactions. The addition of bismaleimide blocked by Diels-Alder reaction enables to postpone the crosslinking to the thermo-molding step and also to substitute disulfides bonds for thioether bonds. The addition of this additive succeeds in doubling the strain at break of gluten-based material but leads to a decrease of the stiffness. The effect of addition of bis- and tetrathiol molecules was also considered as tests. These additives lead to increase by 1.5 times the elasticity of gluten-based materials. A multi-scale study (molecular scale by FTIR, macromolecular scale by SEC and macroscopic scale by tensile test, all supported by DTMA analysis) of the structure and properties showed that a predominant conformation with α-helices which is the case of native gluten, leads to a decrease of elasticity. The formation of β-sheets interproteic interactions was identified as the only responsible of elasticity increases of the material. The formation of proteic aggregates with disulfide and thioeter bonds only plays a secondary role. A reaction mechanism highlighting the conditions that ensure the participation of all types of gluten proteins in the gluten network upbuilding is discussed. Two new promising ways of rubber melt and copolymerization by “grafting from” technique were explored and need to be further developed.