No description available.

Ultraviolet (UV) radiation has been demonstrated in numerous studies to be a major risk factor for non-melanoma skin cancer development. Despite the emergence of current UV-preventative strategies, such as sunscreens and skin-protective clothing, the incidence of non-melanoma skin cancer has continued to rise. This has encouraged investigations on alternative methods for UV prevention. In particular, bovine milk sphingomyelin has been studied for its potential in protecting human skin against UV photodamage. While the previous studies have suggested that sphingomyelin exhibits UV-protective properties in a human skin equivalent model, the exact mechanisms behind sphingomyelin’s photoprotective effects are yet unknown. This thesis aims to further investigate the UV-protective effects of sphingomyelin in normal human epidermal keratinocytes, using nuclear p21 expression as a marker for UV photodamage. Keratinocytes were incubated for 24 hours in a 0.1% sphingomyelin solution and then exposed to 40mJ/cm2 of 302nm UV radiation. After 24 hours of post-UV incubation, nuclear p21 expression was evaluated using immunofluorescence. Confocal images were analyzed for their mean nuclear p21 fluorescence intensity measured in grayscale (0-255). Keratinocytes treated with sphingomyelin showed approximately a 50% decrease in UV-induced mean nuclear p21 intensity compared to keratinocytes with no sphingomyelin treatment (via Tukey’s test; p

A obesidade, doença multifatorial, ocasiona inúmeros distúrbios no metabolismo lipídico e energético, provocando disfunção na bioenergética mitocondrial. A partir deste fato, o presente estudo teve como hipóteses que: o desequilíbrio na bioenergética mitocondrial e as alterações metabólicas causadas pela obesidade são terapeuticamente modificados com o treinamento físico. Dessa maneira, o objetivo do estudo foi avaliar a capacidade oxidativa e conteúdo mitocondrial em tecido adiposo branco, marcadores de doenças cardiosculares (esfingolipídios e N-óxido de trimetilamina, TMAO) e as alterações na composição corporal, desempenho físico e taxa metabólica de repouso (TMR) de mulheres com obesidade submetidas a treinamento físico combinado. A casuística do presente trabalho foi composta de 14 mulheres adultas jovens com diagnótico clínico de obesidade (IMC 33±3 kg/m² e idade 35±6 anos). Foram submetidas a um programa de treinamento físico combinado (exercícios aeróbios e força alternadamente, 55 min à 75-90% da frequência cardíaca máxima, 3 vezes por semana, durante 8 semanas). Todas as participantes foram avaliadas antes e após a intervenção com o treinamento, quanto a: composição corporal, TMR, oxidação de substratos (carboidrato e lipídios) e coeficiente respiratório (QR), desempenho físico, capacidade oxidativa (respiração acoplada: VADP/VOLIGO, e respiração desacoplada: VOLIGO/VCCCP) e conteúdo mitocondrial (enzima citrato sintase, CS) em tecido adiposo branco, nível de esfingolípidios, TMAO e precursores plasmáticos. Os dados foram analisados pelo test t pareado ou Wilcoxon (as pacientes foram consideradas controle de si próprio), após determinação da normalidade da amostra, considerado nível de significância p<= 0,05. Após a intervenção (treinamento físico combinado), houve o aumento da TMR, oxidação de lipídios e desempenho físico, com redução da oxidação de carboidratos e QR, mas não houve perda de peso e alteração da composição corporal. Após o treinamento combinado houve, o aumento da atividade da enzima CS (marcador de conteúdo mitocondrial) e redução à respiração desacoplada (VOLIGO/VCCCP). No plasma, o treinamento físico foi capaz de reduzir os níveis de esfingolipídios e TMAO (fatores de risco cardiovasculares). Também foram encontradas correlações positivas entre TMR, oxidação de lipídios e desempenho físico com CS e negativamente correlacionado com respiração desacoplada. Concluindo, o treinamento físico em mulheres com obesidade aumentou o metabolismo energético, com aumento da TMR, conteúdo e grau de acoplamento mitocondrial, aumentou o desempenho físico e reduziu fatores de risco cardiovasculares (TMAO), independente da perda de peso. / The obesity, a multifactorial disease, causes various metabolic disorders in lipid and energy metabolism, may induce mitochondrial bioenergetic dysfunction. From this, the present study hypothesized that: mitochondrial bioenergetics dysfunction and metabolic problems caused by obesity are be therapeutically modified with physical training. Thus, the objective of study was to evaluated: the oxidative capacity and mitochondrial content in white adipose tissue, markers of cardiovascular diseases (sphingolipids and trimethylamine N-oxide, TMAO) and changes of body composition, physical performance and resting metabolic rate (TMR) of obese women submitted to combined physical training. The present study was composed of 14 young women with obesity (BMI 33 ± 3 kg/m² and age 35 ± 6 years old). They underwent a combined physical training program (aerobic exercises and strength alternately, 55 min at 75-90% of maximal heart rate, 3 times a week, for 8 weeks). All participants were evaluated before and after the intervention: body composition, TMR, substrates oxidation (carbohydrate and lipids) and respiratory coefficient (RQ), physical performance, oxidative capacity (by mitochondrial respiration - Couple: VADP/VOLIGO; Uncoupling: VOLIGO/VCCCP) and Citrate Sinthase activity in white adipose tissue, level of sphingolipids, TMAO and precursors from plasma. Data analysis were made by paired t test or Wilcoxon, after normality determination of the sample, with level of significance p <0.05. After intervention with combined physical training, there was an increase in TMR, lipid oxidation and physical performance, reduced carbohydrate oxidation and RQ, but did not cause weight loss and changes of body composition. In adipose tissue, physical activity increased CS activity (mitochondrial content marker) and reduced uncoupling respiration (VOLIGO/VCCCP). In plasma, physical training was able to reduce levels of sphingolipids and TMAO (cardiovascular risk factors). In addition, positive correlations were found between, TMR, lipid oxidation and physical performance with CS and negatively correlation with uncoupling respiration. Therefore, physical training in obese women improve energy metabolism, with increased TMR, content and degree of mitochondrial coupling, increased physical performance and reduced cardiovascular risk factors, regardless of weight loss.

<p>This thesis covers a wide range of analytical method development for lipid analysis in complex biological samples; from sample preparation using pressurized fluid extraction (PFE) and separation with reversed phase capillary liquid chromatography (RP-LC) to detection by electrospray ionization mass spectrometry (ESI/MS) and tandem MS.</p><p>The requirements for fast, reliable and selective extraction methods with minimal usage of solvents have accelerated the development of new extraction techniques. PFE is one of the new automated, fast and efficient liquid extraction techniques which use elevated temperature and pressure with standard liquid solvents. In this thesis the reliability and efficiency of the PFE technique was investigated for the extraction of total lipid content from cod, herring muscle and human brain tissue as well as for pesticides from fatty foodstuffs. Improved or comparable efficiencies were achieved with reduced time and solvent consumption as compared to traditional methods. </p><p>A RP-LC coupled online to ESI/MS for the analysis of phosphatidylcholine (PC) and sphingomyelin (SM) molecular species was developed and used for the analysis of brain lipids from eight groups of mice treated with vehicle and various neuroleptics. The effect of postnatal iron administration in lipid composition and behavior was investigated. Whether or not these effects could be altered by subchronic administration of the neuroleptics (clozapine and haloperidol) were examined. The results support the hypothesis that an association between psychiatric disorders, behavior abnormalities and lipid membrane constitution in the brain exists.</p><p>Finally, a tandem MS precursor ion scan was used to analyze the developmental profile of brain sulfatide accumulation in arylsulfatase A (ASA) deficient (ASA -/-) as compared to wild type control (ASA +/+) mice. The ASA -/- mice were developed as a model of the monogenic disease metachromatic leukodystrophy with an established deficiency of the lysosomal enzyme ASA. The results showed that an alteration in the composition of sulfatide molecular species was observed between the ASA -/- and ASA +/+ mice.</p><p>This thesis shows that modern analytical methods can provide new insights in the extraction and analysis of lipids from complex biological samples.</p>

This thesis covers a wide range of analytical method development for lipid analysis in complex biological samples; from sample preparation using pressurized fluid extraction (PFE) and separation with reversed phase capillary liquid chromatography (RP-LC) to detection by electrospray ionization mass spectrometry (ESI/MS) and tandem MS. The requirements for fast, reliable and selective extraction methods with minimal usage of solvents have accelerated the development of new extraction techniques. PFE is one of the new automated, fast and efficient liquid extraction techniques which use elevated temperature and pressure with standard liquid solvents. In this thesis the reliability and efficiency of the PFE technique was investigated for the extraction of total lipid content from cod, herring muscle and human brain tissue as well as for pesticides from fatty foodstuffs. Improved or comparable efficiencies were achieved with reduced time and solvent consumption as compared to traditional methods. A RP-LC coupled online to ESI/MS for the analysis of phosphatidylcholine (PC) and sphingomyelin (SM) molecular species was developed and used for the analysis of brain lipids from eight groups of mice treated with vehicle and various neuroleptics. The effect of postnatal iron administration in lipid composition and behavior was investigated. Whether or not these effects could be altered by subchronic administration of the neuroleptics (clozapine and haloperidol) were examined. The results support the hypothesis that an association between psychiatric disorders, behavior abnormalities and lipid membrane constitution in the brain exists. Finally, a tandem MS precursor ion scan was used to analyze the developmental profile of brain sulfatide accumulation in arylsulfatase A (ASA) deficient (ASA -/-) as compared to wild type control (ASA +/+) mice. The ASA -/- mice were developed as a model of the monogenic disease metachromatic leukodystrophy with an established deficiency of the lysosomal enzyme ASA. The results showed that an alteration in the composition of sulfatide molecular species was observed between the ASA -/- and ASA +/+ mice. This thesis shows that modern analytical methods can provide new insights in the extraction and analysis of lipids from complex biological samples.

In Chapter I is a general description of novel metal complexes which hydrolytically cleave peptides, proteins, DNA, and other biological molecules. These reagents are becoming the more important as potential therapeutic agents. A panel of ligands was investigated for coordination to ZrIV and other metals in groups 4, 5, and 6 to effect the greatest degree of hydrolysis. Chapter II describes a ZrIV complex which is capable of hydrolyzing a 30 amino acid peptide, insulin chain B, with amino acid specificity. Oxidized insulin chain B peptide was hydrolyzed after only 4 h of treatment at pH 7.0 and 60 °C using ZrCl4 in the presence of 4,13-diaza-18-crown-6. MALDI-TOF and ESI LC-MS mass spectra indicated that insulin chain B was hydrolyzed by ZrIV at the Gly8-Ser9, Ser9-His10, and Gly20-Glu21 amide bonds within the oligopeptide. To our surprise, the cysteine sulfonic acid sequences Cys(SO3H)7-Gly8 and Cys(SO3H)19-Gly20 were also cleaved. To the best of our knowledge, this constitutes the first example of metal-assisted hydrolysis of a Cys(SO3H)-Xaa amide bond. This is significant in light of the fact that cysteine sulfonic acid formation in proteins is triggered by oxidative stress and has been associated with amyloid fibril formation, Parkinson’s disease, and other deleterious, physiological processes. Chapter III describes the metal-assisted hydrolysis of sphingomyelin which is a principle phospholipid component of animal cell membranes. The sphingomyelin assays showed evidence of metal-assisted hydrolysis after 20 h of treatment at lysosomal pH 4.8 and cytosolic pH 7.0 at both physiological temperature 37 °C and 60 °C. The metal ion CeIV was the most reactive, followed by ZrIV, and then HfIV. The goal of this work is to develop metal-based reagents to reverse the lethal build-up of sphingomyelin that occurs in lysosomes of patients suffering from Niemann-Pick disease.

No description available.

Im Rahmen dieser Arbeit wurden fluoreszierende Sphingomyelin-Analoga zu Studium der sauren Sphingomyelinase (ASM) synthetisiert. Ausgehend von L-Serin wurde ein Sphingosin-Derivat mit natürlicher Stereochemie dargestellt. Anschließend wurde mittels Phosphorodichloridat-Chemie eine Aminoethylphosphat-Gruppe installiert. Zweifache Fluoreszenzmarkierung ergab Sonden mit der Fähigkeit zu Förster-Resonanzenergietransfer (FRET). Diese wurden als Substrate der ASM akzeptiert und erlaubten die Verfolgung der Enzymaktivität in vitro. Durch die Analyse der photophysikalischen Eigenschaften der Fluorophore wurde das allgemeine Konzept der Phasentrennungs-gestützten Signalverstärkung (PS) abgeleitet. Dieses Konzept wurde erfolgreich bestätigt durch die Synthese einer 30-mal leistungsfähigeren zweiten Generation der FRET-Sonde. Ein homogener Assay wurde entwickelt, der die Quantifizierung der ASM-Aktivität erlaubte. Unter Verwendung von gereinigter rekombinanter humaner ASM, HeLa-Zelllysaten oder Lysaten von murinen embryonalen Fibroblasten (MEFs) als Enzymquelle wurde ausschließlich unter den von der ASM bevorzugten Bedingungen eine vollständige und spezifische Hydrolyse der Sonde beobachtet. Des Weiteren erlaubte die Sonde die Detektion relativer Unterschiede der Aktivität der ASM in kultivierten MEFs mittels Fluoreszenzmikroskopie mit Zweiphotonenanregung (2PE). / Fluorescent sphingomyelin analogues have been synthesized to probe the acid sphingomyelinase (ASM). Starting from L-serine, a sphingosine with natural stereochemistry was synthesized. Subsequently, phosphorodichloridate chemistry was used to install an aminoethyl phosphate moiety. Dual fluorescent labeling afforded probes capable of Förster resonance energy transfer (FRET). They were recognized as substrates of ASM and allowed for monitoring of the enzyme’s activity in vitro. Through analysis of the fluorophores’ photophysical properties, the general concept of partition aided amplification of a FRET probe’s signal (PS) was developed. This concept was successfully confirmed by the synthesis of a second-generation probe with 30-fold improved response. A homogenous assay was developed, which allowed for a quantitation of ASM activity. Using either purified recombinant human ASM, or lysates of HeLa cells or mouse embryonic fibroblasts (MEFs) as an enzyme source, complete and specific cleavage was observed exclusively under conditions preferred by ASM. Furthermore, the probe enabled the detection of relative levels of ASM activity in cultivated MEFs using fluorescence microscopy with two-photon excitation (2PE).

Ceramid, ein zentrales Signalmolekül des Sphingolipidstoffwechsels, ist neben der de novo Synthese über die enzymatische Spaltung von Sphingomyelin und Glucosylceramid zugänglich. Genetische Mutationen, die eine Fehlfaltung der verantwortlichen Enzyme saure Sphingomyelinase (aSMase) und Glucocerebrosidase (GCase) begünstigen, könnten somit zu einer Dysregulation des gesamten Sphingolipidstoffwechsels und den damit verbundenen Signaltransduktionsprozessen führen. Niedermolekulare Inhibitoren können in Zellstudien einen Einblick in diese Prozesse geben und den Defekt eines Enzyms simulieren oder eine etwaige Überaktivität derselben Enzyme verhindern. Für derartige Studien ist die Möglichkeit einer zeitaufgelösten Inhibition von Vorteil. Für diese Methode müssten photolabile Schutzgruppen in eine bereits bekannte Inhibitorstruktur integriert werden. Im Fall der aSMase würden sich hierfür myo-Inosit-bisphosphat-Derivate anbieten, die starke, kompetitive Inhibitoren des Enzyms darstellen. Auf dieser Grundlage werden in der vorliegenden Arbeit die Synthese sowie die in vitro und in cellulo Wirkung des ersten zellpermeablen, photoaktivierbaren Inhibitors für die aSMase präsentiert. Kompetitive Inhibitoren können ebenso als sogenannte pharmakologische Chaperone fungieren, welche Proteine durch Herabsetzung der freien Energie des jeweiligen Faltungszustandes stabilisieren. Dies ist besonders bei von Mutationen betroffenen lysosomalen Enzymen von Interesse, um diese vor einem proteasomalen Abbau zu bewahren und einen geregelten Transport in die Lysosomen zu gewährleisten. So wurden in der vorliegenden Arbeit verschiedene myo-Inositderivate als potenzielle pharmakologische Chaperone für die aSMase und GCase synthetisiert. Um eine Verdrängung der Verbindungen vom aktiven Zentrum des Enzyms durch das natürliche Substrat zu beschleunigen, wurde eine Orthoesterfunktion in die Seitenkette der Inhibitorstruktur integriert, die im sauren Milieu der Lysosomen gespalten werden kann. / Ceramide, a central signaling molecule in sphingolipid metabolism, is in addition to the novo synthesis accessible via the enzymatic cleavage of sphingomyelin and glucosylceramide. Genetic mutations that promote misfolding of the responsible enzymes acid sphingomyelinase (aSMase) and glucocerebrosidase (GCase) could thus lead to a dysregulation of the entire sphingolipid metabolism and the associated signal transduction processes. Small molecule inhibitors can provide insight into these processes in cell studies and simulate the defect of an enzyme or prevent eventual overactivity of the same enzyme. For such studies, the possibility of a time-resolved inhibition would be advantageous. For this method, photolabile protecting groups would have to be integrated into the structure of a known inhibitor. In the case of aSMase, myo-inositol-diphosphate derivatives, which represent strong, competitive inhibitors of the enzyme, would be suitable for this purpose. On this basis, the synthesis as well as the in vitro and in cellulo effects of the first cell-permeable photocaged inhibitor for acid sphingomyelinase are presented in this work. Competitive inhibitors can also act as so-called pharmacological chaperones, which stabilize proteins by reducing the free energy of the respective folding state. This is of particular interest in the case of lysosomal enzymes affected by mutations, in order to protect them from proteasomal degradation and to ensure regulated transport into the lysosomes. In the present work, various myo-inositol derivatives were synthesized as potential pharmacological chaperones for aSMase and GCase. To accelerate displacement of the compounds from the enzyme's active site by the natural substrate, an orthoester function was integrated into the side chain of the inhibitor structure, which can be cleaved in the acidic environment of the lysosome.

El ácido 2-hidroxioleico (2OHOA) es un fármaco antitumoral diseñado para regular la estructura y composición de los lípidos de membrana y la función de importantes proteínas de membrana. El objetivo principal de este trabajo fue estudiar cómo el 2OHOA modula la composición lipídica y la estructura de membrana en las células tumorales. Se observó que el 2OHOA indujo profundas alteraciones en el contenido de fosfolípidos, aumentando el contenido de esfingomielina y disminuyendo el contenido de fosfatidiletanolamina y fosfatidilcolina. Este efecto fue específico contra las células cancerosas, ya que el tratamiento no afectó la composición lipídica de las células no tumorales MRC-5 de fibroblastos humanos. El aumento de SM se debió a una activación rápida y específica de las SM sintasas. Como consecuencia de la activación sostenida de la SMS, todo el metabolismo de los esfingolípidos se vio afectado. Finalmente, se evaluó el impacto de todos estos cambios sobre las propiedades biofísicas de membrana mediante espectroscopia de fluorescencia / 2-Hydroxyoleic acid (2OHOA) is a potent antitumor drug that was designed to regulate membrane lipid composition and structure and the function of important membrane proteins. The main goal of this work was to study how 2OHOA modulates the membrane lipid composition and structure of tumor cells. 2OHOA induced dramatic alterations in phospholipid content, increasing sphingomyelin mass, and decreasing phosphatidyl-ethanolamine and phosphatidylcholine. This effect was specific against cancer cells as it did not affect non-tumor MRC-5 cells. The increased SM mass was due to a rapid and highly specific activation of SM synthases. As a consequence of the sustained activation of SMS, the whole sphingolipid metabolism was affected. Then, the impact of all these changes on membrane biophysical properties was evaluated by fluorescence spectroscopy