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L’inhibition ou l’inactivation de la myostatine (mstn) entraine une hypertrophie musculaire qui permet d’envisager des thérapies efficaces dans la lutte contre la fonte musculaire dans de nombreuses pathologies (myopathies, maladies chroniques, sarcopénie). Cependant, le muscle déficient en mstn présente une fatigabilité musculaire accrue, associée à des altérations du métabolisme mitochondrial et lipidique. Or, les membranes musculaires et mitochondriales sont constituées principalement de lipides et phospholipides. Ces derniers participent au maintien de la structure et de la fonction métabolique de la fibre. Ils interviennent également dans la chaine respiratoire jouant un rôle clé dans la bioénergétique mitochondrial. Dans ce travail de thèse, nous avons émis l’hypothèse que la composition lipidique musculaire et mitochondriale est altérée dans le muscle KO mstn, expliquant en partie les altérations métaboliques et fonctionnelles de ce phénotype. Dans un second temps, nous avons recherché si l’entrainement en endurance normalise ces altérations phénotypiques musculaires. Nos résultats ont montré dans le muscle KO mstn une diminution de l’expression des différents transporteurs membranaires des lipides (FAT/CD36, FABP3, FATP1 et FATP4) associé à une réduction de l’activité des enzymes impliquées dans l’oxydation lipidique (Citrate synthase et βHAD) et une diminution de la lipogenèse (chute du contenu en triglycérides et en acides gras libres). D’une manière intéressante, nos résultats montrent une diminution de la proportion en cardiolipide au niveau de la membrane mitochondriale, en relation avec une réduction de l’expression des gènes PGPS et CRLS1, impliqués dans le processus de synthèse de cardiolipide. Nous avons également établi que 4 semaines d’entrainement en endurance sur tapis roulant améliorent en particulier la performance aérobie des souris KO mstn, qui retrouvent une capacité d’endurance comparable à celle des souris contrôles entrainées. L’expression des marqueurs de l’oxydation lipidique et du métabolisme oxydatif est également améliorée (Cpt1, Pparδ, Fas, contenu mitochondrial et citrate synthase). L’entraînement permet aussi d’augmenter l’activité des enzymes mitochondriales et la proportion membranaire en cardiolipide uniquement chez les souris KO mstn. En conclusion, ces résultats suggèrent que les qualités oxydatives du muscle hypertrophié KO mstn peuvent être remodelées sans impacter l’effet bénéfique hypertrophique. Enfin, ils présentent le métabolisme lié au cardiolipide et lipidique de manière générale comme de nouvelles pistes à explorer pour améliorer le métabolisme du muscle KO mstn et sa fonction mitochondriale. / Myostatin (mstn) inactivation or inhibition is considered as a promising treatment for various muscle-wasting disorders because it promotes muscle growth. However, mstn-deficient hypertrophic muscles show strong fatigability associated with abnormal mitochondria and lipid metabolism. Muscle membrane maintains the structure and the metabolic function of the fibre, and mitochondrial membrane including respiratory chain complexes, are composed mainly of lipids and phospholipids playing functional role in mitochondrial bioenergetics. In our study, we hypothesized first that changes in the muscle and mitochondrial lipid composition could exist in the KO mstn muscle, in relation with the metabolic and functional alterations, secondly that endurance training can normalize these phenotypic muscle alterations. We reported in KO mstn muscles a decrease of fat membrane transporter levels (FAT/CD36, FABP3, FATP1 and FATP4) associated with decreased lipid oxidative pathway (citrate synthase and βHAD activities) and decreased lipogenesis (decreased triglyceride and free fatty acids content). Interestingly, we demonstrated a decrease in mitochondrial cardiolipin content, in relation with a decrease in PGPS and CRLS1 gene expressions. Then, we showed in KO mstn mice that 4 weeks of daily running exercise session (65-70% of the maximal aerobic speed for 1 hour) improved significantly aerobic performance, particularly the endurance to levels comparable to those of trained wild type littermates.The expression of oxidative and lipid metabolism markers also was increased, as indicated by the upregulation of the Cpt1, Ppar, Fas genes, and increased citrate synthase level and mitochondrial protein content in KO mstn muscle. Interestingly, mitochondrial enzyme activity and the cardiolipin fraction in the mitochondrial membrane are increased by training only in KO mstn mice. In conclusion, these results suggest that the combination of mstn inhibition and endurance training could increase the muscle mass while preserving the physical performance. In addition, cardiolipin and lipid-related pathways could represent new targets to improve mstn-deficient muscle metabolism and restore mitochondrial function.

Palicourea marcgravii St. Hil. (Rubiaceae) vulgarmente conhecida como \"erva de rato\" é uma planta tóxica brasileira de grande interesse econômico na pecuária, por provocar, em bovinos, convulsões, arritmias cardíacas e \"morte súbita\" em elevado número de animais. No presente trabalho, as folhas dessecadas e moídas de P. marcgravii foram extraídas com etanol 95 %, a temperatura ambiente, por percolação; o extrato resultante foi fracionado por partição com acetato de etila e butanol saturado com água. Os resíduos obtidos foram testados \"per os\" em ratos, buscando-se investigar nos mesmos, a presença do princípio ativo tóxico. O resíduo aquoso foi o único que produziu convulsões e morte dos animais. A existência do monofluoroacetato (MF A) no resíduo aquoso foi comprovada biologicamente através do paralelismo entre as retas refetentes às funções dose/porcentagem de letalidade e dose/latência para a 1ª convulsão que foram construídas para o resíduo aquoso e padrão de referência (monofluoroacetato de sódio). O MFA foi também identificado quimicamente através da RMN19F e da cromatografia em camada delgada. Os resultados obtidos comprovaram a presença de MF A nas folhas de P.marcgravii, responsabilizando-o pelos efeitos tóxicos produzidos. Também foram levantadas algumas hipóteses na tentativa de explicar a sintomatologia dos animais intoxicados. / Palicourea marcgravii St. Hil. (Rubiaceae) is one of the most economica1ly important poisonous plants for Brazilian livestock, since it induces not only seizures, cardiac arrythmias, but also \"sudden death\" of large number of the intoxicated animals. ln the present paper, the dissected and grounded leaves of P.marcgravii were extracted with ethanol 95 %, at room temperature, by percolation; the resultant extract was fractionized by partition in ethyl acetate and butanol saturated with water. The obtained residues were administered \"per os\" to rats, trying to look for the presence of the active toxic principIe. The aqueous residue was the only that induced seizures and death to the animals. The presence of monofluoroacetate (MFA) in the aqueous residue was biologica1ly confirmed by the correlation shown in the sodium monofluoroacetate and the aqueous residue dose-response and dose effect curves. MFA was also identified through NMR19F and thin layer chromatography. The obtained results confirmed the presence of MF A in the P.marcgravii leaves; they also suggest that the toxic effects induced by this plant are a consequence of the presence of MFA in its leaves. Further, some hypothesis were also perfomed, in an attempt to better explain the symptoms induced by the plant leaves.

A atual Política Nacional de Plantas Medicinais e Fitoterápicos prevê investimentos em pesquisa e desenvolvimento para suprir a necessidade da indústria nacional em adquirir a matéria prima para a produção de novos fitoterápicos de maneira segura e controlada. As espécies do gênero Smilax, conhecidas popularmente como salsaparrilha, são empregadas na medicina popular como fortificante, antirreumático e antissifilítico e são vendidas em farmácias, casa de ervas e mercados públicos sem que exista um controle de qualidade de sua origem e eficácia. Além disso, a procedência do material é baseada principalmente no extrativismo. O controle de qualidade das drogas vegetais deveria ter uma base mais segura de identificação das drogas através da caracterização e definição de particularidades anatômicas e químicas. Este estudo teve como objetivo apresentar a quantidade, o valor, o modo de preparo, utilização e a procedência da salsaparrilha comercializada no Estado de São Paulo, bem como analisar a anatomia e o perfil químico de 44 amostras de salsaparrilha comercializada. Amostras de raiz foram submetidas às técnicas convencionais de microscopia de luz e eletrônica de varredura. Também foram realizados testes histoquímicos. Para determinar o perfil químico por Cromatografia em Camada delgada (CCD), foram realizados extratos etanólicos de amostras de raízes. O perfil químico do material comercializado foi comparado com o perfil das espécies de Smilax previamente identificadas (S. goyazana A. De Candolle, S. rufescens Grisebach, S. brasiliensis Sprengel, S. campestris Grisebach, S. cissoides Martius ex Grisebach, S. fluminensis Steudel, S. oblongifolia Pohl ex Grisebach e S. polyantha Grisebach). A quantidade média de salsaparrilha comercializada nas farmácias (400g) e nas casas de ervas (20Kg) é elevada se for considerado o fato de que as raízes não são procedentes de cultivo. Embora tenha sido observada grande semelhança entre a estrutura anatômica das amostras de salsaparrilha comercializadas e a estrutura já descrita na literatura para as espécies de Smilax, houve diferenças em relação à organização do floema, à presença de séries de idioblastos contendo ráfides na medula, à ausência de idioblastos fenólicos na medula e presença de metaxilema no centro da estrutura. O teste histoquímico confirmou a presença de amido em todas as amostras comerciais. As análises em CCD dos extratos etanólicos das amostras comerciais evidenciaram diversas manchas com tonalidades variando de amarelo a verde. Além disso, as manchas apresentaram componentes de mesmo Fator de retenção (Rf), indicando semelhança química entre as diversas amostras. No entanto, o padrão de distribuição de manchas, bem como o Rf das amostras comerciais diferiu daqueles obtidos para as oito espécies de Smilax, os quais foram muito similares entre si. / The current National Policy on Herbal Medicine provides investments in drug research and development to supply the need of national industry to acquire the raw material for production new herbal medicines in a safe and controlled way. The species of Smilax, popularly known as sarsaparilla, are used in folk medicine to be effective as tonic, antirheumatic and antisyphilitic properties and they are commercialized in pharmaceutical stores, natural products stores and public markets in Brazil with no quality control of its origin and efficacy. In addition, these plants are still harvested in extractive way. The quality control of herbal drugs should have more secure identification of drugs through the characterization and definition of anatomical and chemical peculiarities. This study aims to present the quantity, value, method of preparation, use and origin of the commercialized sarsaparilla in cities from the São Paulo state and to investigate the anatomy and chemical profile of 44 samples of the commercialized sarsaparilla. Root samples were processed and analyzed using conventional light and scanning electron microscopy techniques. Usual histochemical tests were also performed. Chemical profiles of samples were analyzed by thin layer chromatography (TLC) using ethanolic extracts of the roots. The chemical profile of the commercialized material was compared with profiles of previously identified species of Smilax (S. goyazana A. De Candolle, S. rufescens Grisebach, S. brasiliensis Sprengel, S. campestris Grisebach, S. cissoides Martius ex Grisebach, S. fluminensis Steudel, S. oblongifolia Pohl ex Grisebach and S. polyantha Grisebach). The average amount of sarsaparilla sold in pharmaceutical stores (400g) and natural products stores (20kg) is high if one considers the fact that the roots are not found under cultivation. Although there was a great similarity between the anatomical structure of commercialized sarsaparilla and the structure already described in literature for the Smilax species, there were found some differences in the organization of the phloem, the occurrence of series of idioblasts containing raphides in the pith, the absence of phenolic idioblasts in the pith and the presence of metaxylem in the center of the organ. The histochemical tests confirmed the presence of starch in all commercialized samples. Chemical profile showed several spots with colors ranging from yellow to green. Moreover, the spots showed the same components retention factor (Rf), indicating chemical similarity between the different samples. However, the distribution pattern of spots, Rf value of commercial samples differed from the eight species of Smilax, which were very similar to each other.

The stem bark of the protected tree species, Curtisia dentata (Burm. f.)C.A.Sm., is one of the most popular plant species harvested and traded at traditional medicine markets in South Africa. The overexploitation of C. dentata trees lead to a “Near Threatened” conservation status and the population trend is portrayed as “declining”. In the KwaZulu-Natal Province of South Africa, C. dentata is completely conservation dependent. This study is not based on drug discovery or toxicological studies, but on the concern that the stem bark of C. dentata trees are harvested, prepared into remedies and consumed as traditional medicine without knowledge regarding the chemical compounds in the stem bark, particularly since the chemical composition of C. dentata stem bark was unknown to date. Phytochemical analyses were firstly conducted to determine the chemical composition of C. dentata stem bark using various solvents and various analytical methods, and secondly, to determine how seasons and regional separation of C. dentata trees affect the chemical profiles of C. dentata stem bark from an environmental and nature conservation perspective. Plants are known to contain numerous chemical compounds. Compounds isolated from a particular plant species are therefore not the only compounds present in that species, and although a plant has proven pharmacological properties, they can still cause harm. Previous studies on C. dentata aimed at validating the plant species as a medicinal plant by examining extracts of the leaves, twigs and stem bark’s potentials against known pathogens and selected cancer cells in vitro and in vivo, and its anti-inflammatory, antioxidant and antiverotoxic properties. Four pentacyclic triterpenoids and one steroidal compound were also previously isolated from C. dentata leaves, however, the leaves are not used in traditional medicines, but were suggested as alternative for stem bark as the harvesting of leaves is less destructive. The efficacy of these compounds as therapeutic agents is, however, compromised by their low solubility in water and thus their potential to penetrate permeating biological membranes. Moreover, in vitro toxicity studies distort the picture of its actual potentials on human health as the whole human metabolome and all its processes, including uptake and phase I and phase II biotransformation are not included. In vivo toxicity studies on mammalian animal species may also not present a true picture of a chemical or extract’s toxic effects on humans as animal metabolisms differ from those of humans. The chemical composition of leaves and stem bark may furthermore also be in contrast to some extent, and therefore chemical compounds were also isolated from C. dentata stem bark in this study. Scientific studies on plant-based medicines generally involve the discovery or identification of compounds that may be beneficial, and which can be exploited in future. Chemical compounds in traditional medicines or other plant-based health products which may cause adverse effects are generally ignored. Moreover, scientific studies that consider that some compounds present in plant extracts may derive from contaminants are equally limited. Traditional plant-based medicines are neither standardized nor regulated in South Africa. Users of traditional plant-based traditional medicines therefore consume uncertain dosages of both beneficial and hazardous substances, as well as contaminants simultaneously. Certain chemical compounds are carcinogens or mutagens or have the ability to accumulate in human tissues. Adverse effects may therefore only manifest after several years of use and will subsequently not be connected to the use of a particular traditional plant-based medicine. The goal of the thesis is therefore to provide a holistic portrayal of the full spectrum of chemical compounds in extracts of C. dentata stem bark and to discuss, where literature is available, the effect(s) each chemical compound may have on human health. Moreover, this thesis investigates variations in chemical composition and concentration in individual trees, seasonal variations and variations in composition and concentrations in the stem bark of C. dentata trees from geographically distinct regions. Most unexpected was that not all C. dentata stem bark samples contained chemical compounds with known beneficial potentials at each sampling date, and that chemical compounds may be region-specific and also tree-specific, which confirms that plants produce secondary metabolites according to the needs of each individual plant. Additional insight into the chemical composition and concentration of C. dentata trees is provided by the distribution profiles of amino acids in C. dentata stem bark. Extreme variations within populations and between geographical areas support the need for the cultivation of C. dentata trees to ensure sustainable production of homogenous material for chemical homogeneity. / Environment Science / PhD. (Environment Science)

This diploma thesis deals with the microbial production of biosurfactants of selected bacterial strains. In order to test the biosurfactant production ability, screening methods were chosen to be able to review the potential of the selected strains to produce biosurfactants. With the scope of the work, 11 bacterial strains, which are used as polyhydroxyalkanoate (PHA) producers, have been tested. The ability to produce biosurfactants was tested in all strains both in complex inoculation and mineral production media. The presence of biosurfactants in Pseudomonas putida was detected on the basis of the results obtained after cultivation in inoculation and production media. The bacteria Pseudomonas fulva was put under more deep study to support their production by cultivation in different types of production media supplemented by different sources of carbon and nitrogen, and the effect of cultivation time was tested as well. Biosurfactants produced by these bacteria were subsequently identified by Fourier transform infrared spectroscopy (FTIR) on the basis of which the substances were identified as rhamnolipids. According to thin-layer chromatography result (TLC), Pseudomonas putida produces a mixture of mono- and dirhamnolipids, with monorhamnolipids being more dominant in our samples.

Les compagnies pétrolières améliorent les propriétés de leurs produits et en particulier des carburants par l’ajout d’additifs. Un large choix de familles d’additifs est disponible, tels que les antioxydants ou les agents antidétonants. Dans ce travail, la chromatographie sur couche mince haute performance (HPTLC) a été utilisée pour quantifier certains additifs dans le gazole sans aucune préparation d’échantillon. L’HPTLC est une technique d’analyse qui est couramment utilisée afin d’analyser et quantifier des composés en mélange. Pour améliorer la détection des polymères et la qualité du signal, des méthodes de dérivatisation ont été utilisées. Afin de confirmer l’identification des composés et obtenir des informations structurales, un couplage direct entre l’HPTLC et la spectrométrie de masse a été développé. Les sources d’ionisation, comme la source DESI(Desorption Electrospray Ionization), la source DART (Direct Analysis in Real Time) et la source MALDI (Matrix Assisted Laser Desorption Ionization) ont été évaluées. Il est apparu que la source MALDI était la plus adaptée pour la désorption des additifs sur plaque HPTLC. Après des essais et optimisations sur différentes phases stationnaires, une méthode HPTLC-MALDI sur phase cellulose a été développée et a permis de détecter les détergents aux teneurs réelles dans le gazole. Parallèlement, l’HPTLC a été couplé pour la première fois à la source ASAP (Atmospheric Solids Analysis Probe). / Oil companies increase the quality of their products such as fuels by using additives. A large variety of additives can be used, such as antioxidants or antiknock agents. In this study, high performance thin layer chromatography (HPTLC) was used to quantify some additive in diesel fuel without sample preparation. HPTLC is an analytical technique used to characterize and quantify compounds in mixtures. To increase polymer detection and signal quality, derivatization methods were used.In order to confirm the analyte identification and to provide structural information, a method based on the direct coupling of HPTLC to mass spectrometry (MS) was developed. Ionization sources such as DESI (desorption electrospray ionization), DART (direct analysis in real time) and MALDI (matrix assisted laser desorption ionization) were evaluated. It appeared that MALDI was the most suitable source to efficiently desorb the additives on HPTLC plate. After several tests and optimizations on different stationary phases and ionization sources, a HPTLC-MALDI method was developed on cellulose and allowed to detect surfactant in diesel fuel at real concentration. At the same time, ASAP (atmospheric solids analysis probe) was coupled for the first time to HPTLC.

(EN) Vernix caseosa is a white creamy substance that covers the skin of a newborn. It is produced during the third trimester by the skin of the baby and remains there until the age of one or even two weeks. It is uniquely human. In utero, vernix protects the skin from maceration, during the birth it serves as a lubricant and after the delivery it protects the baby against infection and regulates the temperature. As vernix is produced in third trimester, prematurely born infants lack it and this may lead to, among other things, suffering from desiccation and therefore heat loss. It is important to study it thoroughly and to find a suitable substitute of vernix for the preterm infants. Vernix consists of lipids, proteins and 80 % water. This project is aimed at the lipids. Vernix is composed of 10 % of lipids. Basic analytical methods of pocessing vernix were searched. The methods of isolation, separation and transesterification have been optimized for the lipids. For separation, thin-layer chromatography has been chosen. The method of the lipid analysis of intact molecules by MALDI-TOF MS has been optimized for these lipids. The results were confirmed using fragmentation spectra and transesterification. Esterified lipids were measured by gas chromatography coupled with mass spectrometry detection....

This dissertation contains the following sections. Chapter 1 contains a detailed description of the theory of thin layer chromatography (TLC). Chapter 2 describes the benefits and practical considerations of elevated temperatures in liquid chromatography (LC). The porous graphitic carbon (PGC) I modified as part of my work is often used in elevated temperature LC. Chapter 3 shows a thermodynamic analysis of chromatographic retention at elevated temperature, and Chapter 4 contains a closer look at the van 't Hoff equation in LC and how it can be used in retention modeling. In Chapter 5, I describe a new procedure for microfabricating TLC plates that avoids the volume/feature distortions that occurred in our first microfabrication. The primary advance of this work was the priming of the carbon nanotube (CNT) forests with chemical vapor deposition (CVD) carbon and atomic layer deposition (ALD) alumina, which permitted effective ALD-like deposition of SiO2. Chapter 6 describes advancements in the microfabrication process of TLC, which excluded the use of the CVD carbon and Al2O3 coating as described in Chapter 5. The use of ozone, to lightly oxidize the CNT surface, primed the material for direct ALD deposition. Chapter 7 gives a detailed surface analysis of the microfabrication process up to and including the CNT forest. It was noticed that a channeling effect was present during Rutherford backscattering analysis of the CNTs. Additionally, characterization of CNTs using time-of-flight secondary ion mass spectrometry in the negative ion mode showed an odd-even effect for a homologous series of carbon, where the even moieties had a stronger signal. Chapter 8 describes the functionalization of PGC with di-tert-amyl peroxide (DTAP) and its effect on increasing the chromatographic performance as seen by a reduction in the tailing factors of test analytes. Chapter 9 -- 13 are detailed X-ray photoelectron analyses of the thin films and CNTs used in producing microfabricated TLC plates.

Desorption Electrospray Ionization Mass spectrometry Imaging (DESI-MSI) is an area of great interest and a promising tool in the field of chemical imaging. It is a powerful, label-free technique, which can determine, map and visualize different molecular compounds on a sample surface. The amount of information acquired in a single DESI-MSI experiment is enormous compared to other techniques, as it can simultaneously detect different compounds with their spatial distribution on the surface. The experiment can be used to produce two-dimensional and three-dimensional images. Chapter 2 focuses on the design and optimization of the setup for performing DESI-MS imaging on various substrates. The proposed setup was tested for its lateral spatial resolution. To provide proof-of-concept of the design, preliminary tests were performed to generate images from commercial thin layer chromatographic plates and photographic paper. Chapter 3 focuses on demonstrating the compatibility of novel microfabricated Thin Layer Chromatography plates (M-TLC plates) for detection with DESI-MSI.