Dissertations / Theses on the topic 'Sulfated'

Chemoenzymatically synthesized low molecular weight lignin polymers have been previously found to be potent inhibitors of a number of serine proteases via allosteric mechanisms targeting heparin binding sites. Herein, we describe the creation of synthetic sulfated β-O4 lignin (SbO4L) polymer, which is more homogenous compared to previous lignins with respect to its inter-monomeric linkage. SbO4L is a selective inhibitor of thrombin and plasmin. SbO4L was found to act via a unique mechanism targeting thrombin exosite 2 in a manner similar to platelet glycoprotein Ibα (GPIbα). Advanced hemostasis and thrombosis assays demonstrated that SbO4L acts via a dual mechanism: as an anticoagulant, by allosteric inhibition of thrombin catalysis; and as an antiplatelet agent, by competing with platelet GPIbα. These mechanisms are comparable in potency to low molecular weight heparins currently used in the market, indicating that targeting exosite 2 may yield clinically useful drugs in the future. Since the β-O4 type lignin was found to be selective for thrombin and plasmin, we hypothesized that other scaffolds from lignins could be potent inhibitors of other serine proteases. In particular, we screened a library of synthetic sulfated small molecules against factor XIa – an emerging target for prophylactic anticoagulation. Our search identified a sulfated benzofuran trimer (a mimic of β-5 type linkage found in lignins) as a potent inhibitor of factor XIa. Surprisingly, this inhibitor did not compete with heparin. A plausible binding site in the A3 domain of factor XIa was proposed by using molecular modeling techniques. The binding pose demonstrated good correlation with the structure activity data from in vitro studies. Further confirmation that the apple domains were required was proved by testing the trimer against recombinant catalytic domain. A 40-fold decrease in activity was observed. A temperature-dependant perrin plot demonstrated that factor XIa undergoes a large conformational change in the presence of the trimer, which is possibly converting the enzyme back into the zymogen-like shape. In general, the synthetic sulfated lignins can act as a useful foundation to develop anticoagulant, antiplatelet, and anti-inflammatory molecules in the future.

Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2013-04-22T14:23:07Z No. of bitstreams: 1 Amália Pereira.pdf: 2389201 bytes, checksum: f4319685be25041470efab01267b4643 (MD5)
Made available in DSpace on 2013-04-22T14:23:07Z (GMT). No. of bitstreams: 1 Amália Pereira.pdf: 2389201 bytes, checksum: f4319685be25041470efab01267b4643 (MD5) Previous issue date: 2004
Catalisadores baseados em óxidos sulfatados possuem diversas aplicações industriais, principalmente em reações que demandam sítios ácidos fortes. Visando a desenvolver métodos para obter óxidos ácidos, neste trabalho se estudou o efeito dos métodos de preparação nas propriedades dos óxidos de zircônio dopado com ferro.Os precursores foram preparados por método sol-gel a partir do nitrato de ferro e oxicloreto de zircônio à temperatura ambiente. Os sólidos secos foram impregnados com uma solução aquosa de ácido sulfúrico (0,25 e 0,5mol.L-1) e calcinados a 500 e 550°C. Foram preparadas amostras com razões molares Fe/Zr=0,2; 0,4 e 0,8, além dos óxidos puros de ferro e zircônio. Os sólidos foram caracterizados por análise química, espectroscopia no infravermelho com transformada de Fourier, difração de raios X, análise térmica (DTA e TG), medida da área superficial específica, redução termoprogramada, medidas de acidez (TPD de amônia) e espectroscopia fotoeletrônica de raios X.Observou-se a formação das fases tetragonal e monoclínica nas amostras à base de óxido de zircônio, enquanto a hematita foi encontrada nas amostras à base de óxido de ferro. Após a sulfatação dos sólidos, a fase monoclínica desapareceu enquanto a fase tetragonal foi estabilizada. Por outro lado, a hematita não foi afetada pelos íons sulfatos. A presença de ferro no óxido de zircônio também estabilizou a fase tetragonal, independente da presença de sulfato. Nas amostras com razão Fe/Zr=0,8, a hematita foi segregada, co-existindo com a fase tetragonal da zircônia. As áreas superficiais específicas dos sólidos aumentaram pela adição de ferro e foram afetadas pelos íons sulfato, mas nenhuma tendência regular foi observada em função das condições de preparação. O óxido de zircônio mostrou maior capacidade em aceitar e estabilizar íons sulfato na estrutura do que o óxido de ferro. O óxido de zircônio puro foi estável em condições redutoras na faixa de 25-1000°C, mas depois da sulfatação os sólidos perderam espécies sulfatos a 500°C. Os óxidos de ferro, entretanto sofreram redução próxima de 350°C independentemente da presença de íons sulfato. A zircônia pura foi mais ácida do que a hematita pura. A adição de ferro na zircônia não afetou significativamente o número de sítios ácidos, exceto no caso da amostra Fe/Zr(molar)=0,2 calcinada 500°C e tratada com solução 0,25mol.L-1 de ácido sulfúrico. Em geral, os sólidos mais ácidos foram produzidos usando-se estas condições. A zircônia sulfatado possui somente sítios ácidos fortes. Entretanto, a adição de ferro gerou sítios de acidez moderada e este efeito aumentou com a quantidade de ferro nos sólidos. O óxido de ferro produziu sítios de diferentes forças ácidas. A composição superficial também variou com a natureza do sólido e com o método de preparação. Os teores de ferro e enxofre foram mais baixos na superfície do que no volume. Observou-se também que o teor de ferro na superfície dos sólidos diminuiu devido à sulfatação. Entretanto, os sólidos com teores mais elevados de ferro foram capazes de reter mais enxofre na superfície. A melhor condição para se obter sólido ácidos à base de zircônio é preparar preparado pela impregnação do hidróxido de zircônio dopado com ferro (Fe/Zr=0,2), tratado com solução 0,25mol.L-1 de ácido sulfúrico seguido da calcinação a 500°C. Este material possui elevada área superficial (223m2/g), sendo promissor para aplicações catalíticas.
Salvador

Sulfated zirconia has attracted extensive attention due to its superactivity to isomerize alkane and alkenes. Platinum or iron/manganese promoted sulfated zirconia has been shown to increase the reaction rate. These catalysts are normally activated to 650-725°C in air before use. Through on-line analysis of evolved gas species during dynamic heating of the catalysts, some significant information can be obtained concerning the activation mechanism. In the present investigation, combined TG-FTIR, TG-MS and TGDTA techniques were utilized to measure the weight loss of the samples, analyze the evolved gas species and to monitor the phase transformation temperature of the solid. Four samples [(1%) 5% Pt/S04 2-VZr02, 2% Fe/0.5% Mn (iron(III)nitrate, maganese(II) nitrate treated/iron(III) sulfate, maganese(II) sulfate treated)/S04 2-/Zr02] were studied by thermal analysis. Also, the catalytic activity of 0.4% Pt/S04 2-/Zr02 was evaluated the isomerization and oligomerization of 1-hexene. It was found that this type of catalyst has high catalytic activity even at ambient and subambient temperatures.

The discovery of heparin in 1916 resulted in a huge impact on the practice of medicine. Heparin has played a major role in alleviating thrombotic disorders and has also exhibited effects on almost every major system in the human body. Over the past few decades, more and more heparin-protein interactions have come to light. It is implicated to modulate several important processes such as cell growth and differentiation, inflammatory response, viral infection mechanism etc. More interesting is the observation that these interactions are considerably specific with regard to oligosaccharide sequences which have specific spatially oriented sulfate groups modulating the responses. However, due to the complex nature of these interactions and lack of effective computational capabilities, predicting these interactions is challenging.An alternative approach to modulating heparin-protein interactions would be to screen a library of molecules having a diverse distribution of the negative charges and screen them against various proteins of interest to obtain valuable information about the binding/selectivity requirements. This approach would not only yield molecules with potential clinical viability, but may also yield molecules that help decipher native mechanisms regulating proteins, which is called chemical biology in today's terms. Since the difficulties associated with carbohydrate synthesis are well known, well characterized highly sulfated oligosaccharide library screening is considered nearly impossible. Thus, the main aim of this project was to develop an effective method for the synthesis of a library of variably sulfated, non-carbohydrate molecules. The library would contain varying in the number of sulfate groups, offer positional variants of the sulfate groups and provide molecules of varying length so as to afford structural diversity necessary to mimic the heparin sequences. Previous attempts in our laboratory to synthesize such a library encountered two major problems: 1) dimerization of polyphenols due to difficult protection / deprotection strategies and 2) ineffective purification of highly water soluble sulfated molecules. To overcome the problem of protection-deprotection, “click” chemistry has been used in this work for dimerization of polyphenols without any protective groups. To overcome the second problem, a non-aqueous method of purification of highly sulfated molecules was developed, which is the first such report.As a proof of concept, a small library of 14 sulfated monomers and dimers and 8 non-sulfated dimers was generated. The protocol for dimerization of free polyphenolic molecules in has been established to use “click” chemistry for coupling the monomers without the need to protect the free hydroxyl groups. Thus by circumventing the inefficient protection-deprotection protocol, there is a tremendous improvement in yields, ease of purification and characterization and greater productivity allowing the synthesis of more number of molecules in a relatively shorter span of time. By masking the charge of the sulfate using an appropriate counter-ion and owing to the inherent lipophilicity of the aromatic scaffold, these highly charged molecules could be purified using normal phase silica gel chromatography. This method reduced the purification time from previous over 48 hours with the aqueous method to approximately 15 minutes. Further, this purification protocol may be possibly automated so as to truly generate a large library of variably sulfated non-carbohydrate molecules for the first time. Screening this library of 22 sulfated and unsulfated molecules against three enzymes of the coagulation cascade – factors IIa, Xa and XIa – has provided a wealth of information with regard to engineering specificity for recognition of these enzymes. The screening led to the identification of CS3 which inhibited factor XIa with an IC 50 of ~ 5 μM and other enzymes with an IC 50 of > 500 μM as a lead candidate with high selectivity. The success of this strategy bodes well for understanding the heparin-protein interactions at a molecular level. Previous attempts in our laboratory to synthesize such a library encountered two major problems: 1) dimerization of polyphenols due to difficult protection / deprotection strategies and 2) ineffective purification of highly water soluble sulfated molecules. To overcome the problem of protection-deprotection, “click” chemistry has been used in this work for dimerization of polyphenols without any protective groups. To overcome the second problem, a non-aqueous method of purification of highly sulfated molecules was developed, which is the first such report.As a proof of concept, a small library of 14 sulfated monomers and dimers and 8 non-sulfated dimers was generated. The protocol for dimerization of free polyphenolic molecules in has been established to use “click” chemistry for coupling the monomers without the need to protect the free hydroxyl groups. Thus by circumventing the inefficient protection-deprotection protocol, there is a tremendous improvement in yields, ease of purification and characterization and greater productivity allowing the synthesis of more number of molecules in a relatively shorter span of time. By masking the charge of the sulfate using an appropriate counter-ion and owing to the inherent lipophilicity of the aromatic scaffold, these highly charged molecules could be purified using normal phase silica gel chromatography. This method reduced the purification time from previous over 48 hours with the aqueous method to approximately 15 minutes. Further, this purification protocol may be possibly automated so as to truly generate a large library of variably sulfated non-carbohydrate molecules for the first time. Screening this library of 22 sulfated and unsulfated molecules against three enzymes of the coagulation cascade – factors IIa, Xa and XIa – has provided a wealth of information with regard to engineering specificity for recognition of these enzymes. The screening led to the identification of CS3 which inhibited factor XIa with an IC 50 of ~ 5 ?M and other enzymes with an IC 50 of > 500 ?M as a lead candidate with high selectivity. The success of this strategy bodes well for understanding the heparin-protein interactions at a molecular level.

Abstract INVESTEGATION OF ANTICOAGULATION PROPERTIES OF SULFATED GLYCOSAMINOGLYCAN MIMETICS By Elsamani Ismail Abdelfadiel, MS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University Virginia Commonwealth University, 2017. Supervisor: Umesh R Desai Professor, Department of Medicinal Chemistry The existence of thrombosis in numerous pathophysiological situations formed a vast necessity for anticoagulation therapy. Thrombin and factor Xa are the only two factors of the entire coagulation cascade that have been major targets for regulation of clotting via the direct and indirect mechanism of inhibition. Our recent discovery of sulfated non-saccharide glycosaminoglycan mimetics, especially G2.2, that demonstrates highly selective cancer stem-like cells (CSCs) inhibition activity. G2.2 inhibited the growth of CSCs from multiple cancer cell lines. To evaluate its in vivo anticoagulation effect, we asked a contract research organization (CRO) to produce 20 g of material, labelled as G2.2Y. Evaluation of G2.2C in HT-29 xenograft mouse model showed a significant reduction in tumor volume and CSC markers, but unexpected bleeding consequences in some animals were observed. Also in a tail bleeding experiment, G2.2Y showed a significant enhancement in bleeding volume. Comparable studies with G2.2 synthesized in our laboratory had shown no bleeding effects. To investigate the difference between the two G2.2 samples (G2.2W (white) and G2.2Y (Yellow) that were performed using UPLC-MS characterization, we were able to determine that the G2.2Y sample was an 85:15 blend of two compounds. Elemental, NMR and MS data revealed that G2.2W was fully sulfated flavonoid derivative, as expected, but G2.2Y contained one less sulfate group. We tested both agents for their inhibition of various coagulation factors and revealed that G2.2Y inhibited fXIa nearly 2-fold better in comparison to G2.2W. Furthermore, activated partial thromboplastin time assay (APTT) indicated that G2.2W exhibited almost 3-4-fold less anticoagulant activity compared to G2.2Y. This indicates that the loss of just one sulfate group could induce substantial side effects and lead to a discovery of new anticoagulant agent. Such structure–activity relationship is important to understand if the in vivo metabolism of the agents leads to accumulation of de-sulfated products.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
FundaÃÃo de Amparo à Pesquisa do Estado do CearÃ
This work reports the preparation and characterization of collagen-sulfated polysaccharide films and applications in coating of cardiovascular prostheses. The films were prepared by adding the sulfated polysaccharide solution on soluble collagen (0, 5, 10, 20 and 25 %). The blends of collagen-sulfated polysaccharide were casted in acrylic molds, and dried at low temperature. The physico-chemical properties of collagen-sulfated polysaccharide films, were studied using infrared spectroscopy that showed absorptions typical of collagen and sulphated polysaccharide; the thermal analysis (DSC) showed that the structural integrity of collagen was unmodified during the extraction process and the polysaccharide present in the samples did not decrease the thermal stability of the collagen, which was higher for the concentrations of 20 %. In the thermal analysis (DTG), the addition of the polysaccharide caused a small decrease of the stability of thermal degradation and when was adding 5 % of the sulfated polysaccharide to the collagen, a reduction in its stability occurs, however, in the concentrations of 10, 20 and 25 %, the stability of thermal degradation of the film increased. In the impedance spectroscopy, the results are associated possibly, to the interactions between the macromolecules. The blood compatibility was analyzed by platelet adhesion and the results showed that the films possess hemocompatibility and antitrombogenic properties.
Este trabalho aborda sobre o preparo e a caracterizaÃÃo de filmes de colÃgeno-polissacarÃdeo sulfatado e sua aplicaÃÃo para revestimento de prÃteses vasculares. Os filmes foram obtidos a partir da mistura de soluÃÃo de colÃgeno e polissacarÃdeo sulfatado, nas concentraÃÃes 0, 5, 10, 20 e 25 % do polissacarÃdeo. As soluÃÃes de colÃgeno-polissacarÃdeo sulfatado foram formatadas em moldes de acrÃlico em condiÃÃes de baixa umidade e temperatura. A caracterizaÃÃo fÃsico-quÃmica dos filmes de colÃgeno-polissacarÃdeo sulfatado foi estudada por espectrometria no infravermelho que mostraram bandas tÃpicas do colÃgeno e bandas tÃpicas do polissacarÃdeo sulfatado; a anÃlise tÃrmica por DSC mostra que a integridade estrutural do colÃgeno foi preservada no processo de extraÃÃo e que a adiÃÃo do polissacarÃdeo nas amostras nÃo diminuiu a estabilidade tÃrmica do colÃgeno tendo sido maior na concentraÃÃo de 20 %. Na anÃlise tÃrmica por DTG, a adiÃÃo do polissacarÃdeo causa um pequeno decrÃscimo da estabilidade de degradaÃÃo tÃrmica e ao adicionarmos 5 % do polissacarÃdeo sulfatado ao colÃgeno, ocorre uma reduÃÃo em sua estabilidade, porÃm, a partir daÃ, nas concentraÃÃes 10, 20 e 25 %, a estabilidade de degradaÃÃo tÃrmica do filme vai aumentando. Na espectroscopia de impedÃncia, os resultados estÃo associados, possivelmente, a interaÃÃes entre as macromolÃculas. A compatibilidade sangÃÃnea foi analisada por adesÃo de plaquetas e os resultados mostraram que os filmes possuem hemocompatibilidade e propriedades antitrombogÃnicas.

The occurrence of thrombosis in several pathophysiological conditions creates a huge need for anticoagulation therapy. Thrombin and factor Xa have been prime targets for regulation of clotting through the direct and indirect mechanism of inhibition. This work investigates chemo-enzymatically prepared oligomers of 4-hydroxycinnamic acids (DHPs) as potential anticoagulants. Oligomers were prepared through peroxidase-catalyzed oxidative coupling of 4-hydroxycinnamic acids. The products resulting from this reaction are called CDs, FDs and SDs. Structurally, these sulfated DHPs are unique and do not resemble any of the anticoagulants known in the literature.DHP oligomers were found to increase clotting times at concentrations comparable to heparin. Studies in blood and plasma show that DHPs possess an anticoagulation profile similar to enoxaparin. To understand the mechanism of action of DHPs, we studied the inhibition of thrombin, FXa, FIXa, and FVIIa in the presence and absence of antithrombin. CDs and FDs display a preference for direct inhibition of thrombin and FXa, and exhibit a high level of specificity over FIXa and FVIIa. In the presence of AT, CDs and FDs displayed weaker inhibition of FXa and thrombin suggesting that binding to AT is a competitive side reaction. SDs exhibited potent inhibition of FXa and thrombin in the absence of antithrombin, but was inactive against FIXa and FVIIa representing the best selectivity among the DHPs. For SDs, inhibition of all the pro-coagulant enzymes favored the antithrombin dependent pathway. Binding studies were performed to determine how CDs directly inhibits thrombin. Competitive binding studies suggest that CDs interacts with exosite II and disrupts the catalytic triad of thrombin. These results indicate that the preferred mechanism of CDs action is exosite II mediated allosteric disruption of thrombin. CDs appears to be the first exosite II mediated DTI and this represents a novel mechanism of inhibitor function. The inhibition characteristics of DHPs are unique and radically different in structure from all the current clinically used anticoagulants. To the best of our knowledge this dual mechanism of anticoagulation and unique binding mode has not been described as yet in literature and represents a novel strategy that our laboratory has discovered.

Methane activation with bromine followed by the condensation of the methyl bromide into higher hydrocarbons or oxygenates is a novel route. However, the selective production of monobrominated methane (CH3Br) at high conversions is a crucial prerequisite. A reaction model was developed according to the kinetic data available in the literature and thoroughly studied to investigate the optimum reactor conditions for selective methane bromination in gas phase. It was concluded that at high methane (>
90%) conversions dibromomethane synthesis was favored at high selectivity (~90%) under the following conditions: T=330 °
C, Br:CH4 = 3. Sulfated zirconia included SBA-15 catalysts were prepared and characterized for the catalytic methane activation via bromination. The SBA-15 sol-gel preparation technique was followed and the zirconium was added during the preparation in the form of ZrOCl2·
8H2O with 5-30 mol % ZrO2 with respect to the SiO2 content simultaneously with the silicon source (TEOS). The catalysts were sulfated in 0.25 M H2SO4 solution. The zirconium contents of the catalysts were determined by elemental analysis and 15 wt. % Zr was determined as the highest amount. XRD analysis showed the crystalline zirconia peaks only for high zirconia loadings (>
25 mol % ZrO2) indicating the good distribution of Zr in silica framework at lower loadings. BET surface areas of the sulfated catalysts are in the range of 313-246 m2/g. The porous structures of the catalysts were determined by TEM pictures, which revealed that the increase in Zr content decreased the long range order of pore structure of SBA-15 in agreement with XRD results. The acidities of the catalysts were determined by 1H MAS NMR experiments. Brø
nsted acidity was identified by a sharp 1H MAS NMR line at 10.6 ppm. The highest acidity was observed at 5.2 wt. % Zr loading according to 1H MAS NMR experiments. 29Si MAS NMR analysis showed the formation of Si-O-X linkages (X=H, Zr). Further characterization of Brø
nsted acidity was performed by FT-IR spectroscopy of adsorbed CO at 82 K. The analysis revealed that the Brø
nsted acidity of sulfated catalysts were similar to the acid strength of the conventional sulfated zirconia. In TPD experiments, the basic molecule isopropylamine (IPAm) was adsorbed and decomposition temperature of IPAm was monitored. The temperature decreased from 340 °
C to 310 °
C in sulfated catalysts, indicating the acidic character of these samples. Catalytic methane bromination reaction tests were performed in a quartz tubular reactor. The results showed that 69% methane conversion was attainable over SZr(25)SBA-15 catalyst at 340 °
C. The liquid 1H NMR measurements of the products revealed that >
99% methyl bromide selectivity was achieved.

Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are important, natural polysaccharides which occur in biological (connective) tissues and have various biotechnological and medical applications. Additionally, there is increasing evidence that chemically (over)sulfated GAGs possess promising properties and are useful as implant coatings. Unfortunately, a detailed characterization of these GAGs is challenging: although mass spectrometry (MS) is one of the most powerful tools to elucidate the structures of (poly)saccharides, MS is not applicable to high mass polysaccharides, but characteristic oligosaccharides are needed. These oligosaccharides are normally generated by enzymatic digestion. However, chemically modified (particularly sulfated) GAGs are extremely refractive to enzymatic digestion. This study focuses on the investigation of the digestibility of GAGs with different degrees of sulfation by bovine testicular hyaluronidase (BTH). It will be shown by using an adapted spectrophotometric assay that all investigated GAGs can be basically digested if the reaction conditions are carefully adjusted. However, the oligosaccharide yield correlates reciprocally with the number of sulfate residues per polymer repeating unit. Finally, matrix-laser desorption and ionization (MALDI) MS will be used to study the released oligosaccharides and their sulfation patterns.

Anticoagulants are used as the first line therapy for management and prevention of thrombotic disorders. Thrombin and factor Xa have been the prime targets for regulation of the coagulation cascade. In this work, a small library of 17 benzofuran derivatives were synthesized and screened against thrombin and factor Xa. The derivatives that displayed inhibitory potential were docked on the exosite-II of factor Xa using a docking protocol that was developed in our research group. These compounds were based on the β-5 structural unit found in the oligomer -'CDSO3‘, which was prepared in our lab and was found to inhibit both thrombin and factor Xa by an exosite-II mediated allosteric disruption of the catalytic triad.The results revealed that these β-5 like derivatives are inhibitory against thrombin and factor Xa, although their potency is weak. Thrombin and factor Xa appear to recognize different structural features suggesting a significant selectivity in recognition. Furthermore, a slight preference for the benzofuran scaffold was observed with factor Xa. Probing the mechanism of inhibition using Michaelis-Menten kinetics reveal that these compounds display uncompetitive inhibition of these proteases and the mechanism of inhibition is allosteric. Docking of these compounds on factor Xa were done using GOLD (Genetic algorithm for ligand docking) and the results, explain the observed inhibition profile. The computed docked poses also give an idea of the residues on the exosite-II of factor Xa critical for inhibition. The molecules studied here are radically different in terms of structure and mechanism of inhibition from any other ligand described in literature. This represents an opportunity to discover novel molecules with a possibly different pharmacological and toxicological profile.

The complex pathobiologic mechanisms of emphysema are not fully understood, leaving this deadly disease without effective pharmacotherapy for a cure. This project hypothesized that the sulfated dehydropolymer of caffeic acid (CDSO3) exhibits Fe2+ chelation-based hypoxia inducible factor-1a (HIF-1a) up-regulatory protective activities against in vitro emphysematous cell death and for in vivo reversal of emphysema induced with SU5416, a vascular endothelial growth factor blocker. Using in vitro chromogenic competitive inhibition assays, CDSO3 was shown to chelate Fe2+ (IC50 of 23 µM), but not Fe3+ ions. The trypan blue exclusion and lactate dehydrogenase assays were then employed to examine the cytoprotective activities of CDSO3 against inflammatory, oxidative, elastolytic, and apoptotic cell death using alveolar macrophages, epithelial and endothelial cells. CDSO3 at 10 µM produced significant protective activities against these emphysematous cell deaths by 50-154 %. These protective effects were opposed by the addition of the HIF-1a inhibitors, CAY10585 and echinomycin, and excess Fe2+, but not Fe3+, ions. Emphysema was then induced in rats following a subcutaneous injection of SU5416 at 20 mg/kg, after which CDSO3 at 60 µg/kg was administered to the lungs 3 times/week for two weeks. Treadmill exercise endurance (EE) was measured to assess the functional impairment, while lung tissues were removed for morphological assessments of alveolar airspace enlargement (MLI) and destruction (DI), as well as to measure protein levels using Western blot. SU5416 significantly impaired EE, MLI, and DI by 81 %, 47 %, and 5-fold, compared to the healthy animals, and these were significantly reversed by CDSO3 by 66, 74, and 87 %. CDSO3 treatment did not change the lung cytoplasmic expression of histone deacetylase 2 (HDAC2), HIF-1a, or a pro-apoptotic marker, BAX. However, induction with SU5416 significantly reduced VEGF expression by 52 % and increased cleaved caspase-3 expression by 1.5-fold, compared to the healthy animals, while CDSO3 normalized the expressions of both proteins in these emphysematous animals. However, when CDSO3 was pre-mixed with excess Fe2+, the reversal activities of CDSO3 were diminished. In conclusion, this study has demonstrated the Fe2+ chelation-based HIF-1a up-regulatory dependent in vitro and in vivo lung repairing efficacies for CDSO3 in emphysema.

Submitted by Maria Medeiros (maria.dilva1@ufcg.edu.br) on 2018-08-17T13:19:35Z No. of bitstreams: 1 CARLOS EDUARDO PEREIRA - TESE (PPGEQ) 2017.pdf: 6340687 bytes, checksum: a5c8d06008238c03161add19b46e0a44 (MD5)
Made available in DSpace on 2018-08-17T13:19:35Z (GMT). No. of bitstreams: 1 CARLOS EDUARDO PEREIRA - TESE (PPGEQ) 2017.pdf: 6340687 bytes, checksum: a5c8d06008238c03161add19b46e0a44 (MD5) Previous issue date: 2017-08-02
A peneira molecular MCM-41 é considerada promissora como suporte para os óxidos metálicos em processo de refino de petróleo, adsorventes e catalisadores. Os catalisadores heterogêneos apresentam grande potencial de viabilizar a produção de biodiesel através da reação de transesterificação. A síntese da peneira molecular MCM-41 foi realizada a partir da água deionizada, brometo de cetiltrimetilamônio, hidróxido de amônio, etanol e ortossilicato de tetraetila. O óxido de zircônia foi obtido pelo método sol-gel a partir do oxicloreto de zircônio com hidróxido de amônio à temperatura ambiente e ativado por calcinação a 550 e 700°C. Em seguida o óxido de zircônia ativado foi sulfatado, seco e calcinado a 400 °C. A zircônia sulfatada foi incorporada a peneira molecular MCM-41 por impregnação via úmida, com diferentes proporções mássicas (10, 20, 30, 40, 50%). Verifica-se que a partir das análises de difração de raio X a formação da peneira molecular MCM-41 confirmou a estrutura hexagonal e a fase mesoporosa. Observou-se formação das fases, tetragonal e monoclínica do óxido de zircônia. Através da espectroscopia de absorção na região do infravermelho com transformada de Fourier foi possível detectar picos referentes a presença de íons sulfatos bidentados ligado a superfície da zircônia. As propriedades texturais apresentaram estruturas com poros bimodais após o processo de incorporação da zircônia sulfatada. As micrografias do óxido de zircônia ativadas a 550 e 700 °C apresentaram tricas em sua superfície antes e após de incorporação da zircônia sulfatada. O potencial catalítico foi avaliado na reação transesterificação do óleo de soja por rota metílica. O catalisador com óxido de zircônia ativado a 700 °C e sulfatado e incorporado a peneira molecular com 40% (em peso) apresentou maior conversão de ésteres metílicos 83,8%. No entanto, esta conversão não especifica o óleo obtido como biodiesel de acordo com a norma da Agência Nacional de Petróleo, Gás Natural e Combustíveis. Assim como o índice de acidez. Porém os resultados de densidade e viscosidade estão de acordo com a especificação estabelecida pelas normas.
The MCM-41 molecular sieve is considered promising as a support for the petroleum refining metal oxides, adsorbents and catalysts. Heterogeneous catalysts have great potential to make viable the production of biodiesel through the transesterification reaction. The synthesis of the MCM-41 molecular sieve was performed from deionized water, cetyltrimethylammonium bromide (CTABr), ammonium hydroxide (NH4OH), ethanol and tetraethyl orthosilicate (TEOS). The zirconium oxide was obtained by the sol-gel method from zirconium oxychloride with ammonium hydroxide at room temperature, the material was activated at 550 and 700 °C, and thereafter, sulphated. The material was then activated using the calcination process at 550 and 700 ° C and sulfated. The sulfation process was carried out with a 0.5 mol.L-1 sulfuric acid solution and allowed to stand for 30 minutes, dried for 12 h at 120 ° C and calcined at 400 ° C. The process of incorporation of ZS into the MCM-41 molecular sieve was done using different mass proportions (10, 20, 30, 40 and 50%) in relation to the mass of the MCM-41 molecular sieve, by wet method. It was verified from the analyzes of X-ray diffraction, the adsorption of nitrogen (BET method) and Fourier transform infrared spectroscopy (FTIR) the crystalline and textural properties which confirmed the molecular sieve obtainment and the presence of the tetragonal and monoclinic phases of the sulfated zirconia in the mesoporous structure. The micrographs of activated zirconium oxide at 550 and 700 °C and sulphated showed dispersed particles with the presence of cracks on its surface, after the incorporation there were no modifications in the structure. The catalytic activity was evaluated by the transesterification reaction of the soybean oil via the methyl route, using all the catalysts which were synthesized. The results showed that the catalyst 50_ZS/MCM-41_550 °C showed a greater conversion of methyl esters of 81.4% with the predominant tetragonal phase. The catalyst with the zirconium oxide activated at 700 °C obtained a conversion of 83.8% to the 40_ZS/MCM-41_700 °C catalyst, with the predominant monoclinic phase. However, the ester content of the oil samples was below the value established by the National Agency of Petroleum, Natural Gas and Fuels (ANP). The density and kinematic viscosity of the catalysts under study X_ZS/MCM-41 were in the range of the established standard. The acidity index was above the specified values, confirmed by the high percentage of free fatty acids in the oil.

Glycosaminoglycans (GAGs) are sulfated polysaccharides that mediate a variety of extracellular interactions. Heparan sulfate (HS) is one of the most prominent GAGs on human cell surfaces. Both endogenous proteins, such as growth factors, and exogenous proteins, such as pathogen surface proteins, recognize and bind GAGs to gain access to human cells. Oligosaccharides and other structural analogs of HS and GAGs have been evaluated for a variety of therapeutic targets including angiogenesis and infectious diseases. Development of compounds to block HS-protein interactions has primarily focused on optimizing the degree and orientation of anionic substituents on a scaffold, to mimic HS structure, but their utility is diminished by their large size and non-specific interactions with many proteins. To overcome these limitations, it has been demonstrated that replacing N-sulfo groups on heparin with non-anionic N-arylacyl groups increased affinity and selectivity for binding different heparin-binding proteins. However, the heparin-derived compounds in that work were heterogeneous polysaccharides. Strategies to obtain small, structurally-defined and lower charge ligands are needed to ultimately obtain specific bind-and-block antagonists of HS-binding proteins. This study addresses these challenges by synthesizing N-arylacyl O-sulfonated aminoglycosides as small molecule, structurally-defined ligands to identify novel structures that selectively bind to HS-binding proteins. This study details development of new HPLC and LC-MS methods to separate, characterize, and purify amphiphilic oligosaccharides. The development of these methods enabled the synthesis of a panel of N-arylacyl O-sulfonated aminoglycosides. The compounds in this panel were screened for affinity and selectivity in binding with HS-binding proteins. This work demonstrates for the first time the selective binding of small amphiphilic oligosaccharides with HS-binding proteins. Significantly, individual compounds demonstrate heparin-like affinity for binding with select HS-binding proteins. Structural differences between the N-arylacyl O-sulfonated aminoglycosides, including changing the aminoglycoside core or the structure of the N-arylacyl moiety, are shown to impart specificity for these compounds to selectively bind different HS-binding proteins.

Sulfated Glycoprotein-1 (SGP-1) secreted by Sertoli cells is a homologue of human prosaposin known to be processed into four 15 kDa saposins (A,B,C and D). Saposins activate lysosomal enzymes which break down membrane glycolipids. The objectives of the present investigation were to examine the developmental expression of SGP-1 and its targeting to lysosomes of Sertoli cells, to characterize the lysosomal form(s) of SGP-1, to examine SGP-1's delivery to Sertoli cells phagosomes and finally to assess the effects of androgens on SGP-1 expression.
SGP-1 exists in two forms, a 65 kDa and a 70 kDa form. Labeling experiments demonstrated that the 65 kDa form of SGP-1 is synthesized first then post-translationally modified to the 70 kDa form. Western blot analysis of purified Sertoli cell lysosomes revealed a 65 kDa protein and 15 kDa proteins. The latter were isolated and shown to be saposins which were possibly derived from the 65 kDa precursor. Immunocytochemical studies using anti SGP-1 antibodies demonstrated that lysosomes deliver SGP-1 or/and saposins to phagosomes containing residual bodies. Thus, besides a secretory route of SGP-1 there appears to be a lysosomal pathway that may play a significant role in the hydrolysis of glycolipids of spermatid membranes present in phagocytosed residual bodies. Permeabilization of purified Sertoli Golgi fractions revealed that the 65 kDa protein is strongly associated with Golgi membrane. This association was resistant to low pH, resistant to excess free mannose 6-phosphate and independent of N-linked glycosylation, but sensitive to EDTA treatment. In contrast, the 70 kDa form of SGP-1 was released from permeabilized Golgi Fractions at pH 7.4 and 5.4 but retained at pH 6.4 indicating that it is a soluble protein with biochemical characteristics of a regulated secretory protein. In vivo treatment with tunicamycin resulted in an increase in the density of lysosomal SGP-1 suggesting that N-linked carbohydrate residues are not essential for the targeting of this protein to the lysosomes of Sertoli cells. In contrast, in the nonciliated cells of the efferent duct, tunicamycin treatment resulted in a significant reduction in the labeling density of lysosomal SGP-1 suggesting a sugar dependent lysosomal transport mechanism.
Thus, this study demonstrates a unique lysosomal form of SGP-1 (65 kDa). The targeting of the 65 kDa form to the lysosomes and eventually to the phagosomes involves an early association with Golgi membranes independent of mannose 6-phosphate receptors. The trafficking of the 70 kDa form of SGP-1 to the tubular lumen may depend on its selective segregation into secretion granules within the Golgi apparatus of Sertoli cells. Finally, the expression of SGP-1 by Sertoli cells is modulated by androgens.

Glycosaminoglycans are known to bind biological mediators thereby modulating their biological activity. Sulfated hyaluronans (sHA) were reported to strongly interact with transforming growth factor (TGF)-β1 leading to impaired bioactivity in fibroblasts. The underlying mechanism is not fully elucidated yet. Examining the interaction of all components of the TGF-β1:receptor complex with sHA by surface plasmon resonance, we could show that highly sulfated HA (sHA3) blocks binding of TGF-β1 to its TGF-β receptor-I (TβR-I) and -II (TβR-II). However, sequential addition of sHA3 to the TβR-II/TGF-β1 complex led to a significantly stronger recruitment of TβR-I compared to a complex lacking sHA3, indicating that the order of binding events is very important. Molecular modeling suggested a possible molecular mechanism in which sHA3 could potentially favor the association of TβR-I when added sequentially. For the first time bioactivity of TGF-β1 in conjunction with sHA was investigated at the receptor level. TβR-I and, furthermore, Smad2 phosphorylation were decreased in the presence of sHA3 indicating the formation of an inactive signaling complex. The results contribute to an improved understanding of the interference of sHA3 with TGF-β1:receptor complex formation and will help to further improve the design of functional biomaterials that interfere with TGF-β1-driven skin fibrosis.

The postnatal changes in the various epithelial cell types of the different regions of the epididymis at the level of electron microscope (EM), and the expression of various secretory proteins has not been well characterized in developing rats. In this context, Epon embedded epididymides at postnatal days 21, 39, 49, and 56 were examined under the light and electron microscope. Furthermore, expression of two secretory proteins, namely sulfated glycoprotein-2 and immobilin, well characterized in adult animals, were immunolocalized using paraffin sections at various postnatal ages i.e. 7, 15, 21, 28, 39, 49, and 56. The latter was performed to examine the influence of testicular products, such as Sertoli cell derived factors (androgen binding protein), luminal and circulating androgens, degenerating germ cells, and spermatozoa appearing at different postnatal ages, on the morphological differentiation of the epithelial cells and expression of these proteins by these cells. (Abstract shortened by UMI.)

Les phénomènes de désactivation des zircones sulfatées (ZS) dopées ou non au manganèse (MnZS; 0,5 et 2,0 % en masse) au cours de la réaction d’isomérisation du n-butane sont étudiés, à 323 ou 373 K, tout au long d´une réaction (mesures in situ) par spectroscopie à réflexion diffuse dans l´ultraviolet visible et proche infra rouge (UV-vis-NIR) et par l’analyse en ligne des gaz de réaction par chromatographie en phase gazeuse. Les catalyseurs « zircone sulfatée » se désactivent en deux étapes distinctes à 373 K : une phase initiale très rapide, et une seconde plus lente. L´étape initiale de la réaction est toujours en discussion, mais plusieurs travaux scientifiques sont en faveur de la déshydrogénation oxydante (ODH). ZS contient très peu de sites actifs, et leur accès peut être bloqué quand certaines espèces y sont adsorbées. Ce travail confirme le double caractère des produits de l´ODH : l´eau et le butène – leur effets positif et négatif sur l´isomérisation du n-butane. L´activité du catalyseur dépend du degré d´hydratation de ce dernier. Les conditions dans lesquelles (milieu gazeux et température) est activée la ZS sont d´une grande importance. De l´eau en excès, à la surface du catalyseur, peut bloquer l´accès des réactifs aux sites actifs ; diminuant ainsi l´activité de la ZS. Il est montré, dans ce travail, que l´eau formée lors des premières heures est responsable de la première étape de désactivation. Comme reporté dans la littérature, le butène est un des produits de l´ODH. La protonation de ce butène par les sites actifs de Brønsted conduit à la formation des ions carbénium sec-butyle /groupes alkoxy adsorbés à la surface. Un mécanisme bimoléculaire faisant intervenir l´alkylation de l´ion carbénium sec-butyle avec une autre oléfine conduit à un réarrangement C-C. Les oléfines, en grande quantité, peuvent aussi être une des raisons de la désactivation du catalyseur. Nous avons montré que l´addition de propène au milieu réactionnel favorise la formation d´oligomères. Le même phénomène est aussi observé lorsque de l´oxygène est ajouté au système. Lors de la combustion, en présence d´oxygène, des espèces carbonées adsorbées à la surface du catalyseur, de l´eau, du dioxyde de carbone et de la chaleur sont produits. Cette chaleur peut favoriser la formation de polymères. Des oligomères absorbant à 370 et 450 nm ont été détectées à la surface de la ZS. Il est prouvé dans ce travail que qu´il existe une corrélation entre leur formation et la seconde étape de désactivation de la ZS. Ces espèces sont donc d’excellentes candidates pour expliquer la seconde phase de désactivation du catalyseur. Il est mis en évidence que les espèces monoénic allylique (absorbant à 295 nm) détectées à la surface de la ZS, au cours de la réaction d´isomérisation du n-butane, ne sont pas responsables de la désactivation du catalyseur, comme reporté dans la littérature, mais seulement des espèces spectatrices. Ces espèces ne sont pas détectées dans les spectres UV-vis de la MnZS bien que ce catalyseur se désactive très vite. Les spectres UV-vis de la MnZS montrent deux bandes. Une attribution erronée de ces bandes dans la littérature a pu être corrigée grâce à des calculs. La bande d’absorption à 580 nm émane du Mn4+ et celle à 680 nm correspond à Mn3+
The deactivation phenomena during n-butane isomerization on sulfated zirconia (SZ) and manganese-promoted sulfated zirconia (MnSZ, 0. 5 or 2 wt% Mn) are investigated at 323 or 373 K by in situ UV-vis-NIR diffuse reflectance spectroscopy and on-line gas chromatography. Sulfated zirconia (SZ) catalysts are found to deactivate in two steps at 373 K: a fast initial and a slow second phase. The well accepted mechanism for the initiation step of the isomerization is the oxidative dehydrogenation (ODH). SZ is an acid catalyst with very few active sites. The access to these sites can be blocked when some species are adsorbed on them. This work confirms the duality effect of the products of ODH: water and butene - their positive and negative effects on n-butane isomerization. An appropriate hydration level of SZ is important for the activity of the catalyst. Thus, the activation conditions (atmosphere and temperature) play a major role. However, excess water on the catalyst surface (band at 1910 nm in the NIR range) can block the access of the reactant to the active sites and thus diminishes the catalyst activity. In this work, it is shown that the water formed during the first few hours on stream is responsible for the first deactivation step. As it has been reported in the literature, butene is another product of ODH and after its protonation by the Brønsted acid sites; sec-butyl carbenium ions stabilized by alkoxy groups are formed. The skeletal rearrangement can proceed through a bimolecular mechanism via an alkylation of the secondary butyl carbenium ion with another olefin. However, the presence of olefin in high quantities can be another reason for the deactivation of the catalyst. The addition of propene to the feed leads to the formation of oligomers. A similar effect on the deactivation of the catalyst is observed when oxygen is present in the system. The addition of oxygen to the feed leads to the combustion of carbonaceous deposits with the formation of water, carbon dioxide and heat. Moreover the heat can favour the formation of polymers. Oligomers were detected on the catalyst surface, with their absorption bands at 370 nm and 450 nm. The experiments showed that their formation corresponds to the second phase in SZ activity decline. These species are good candidates to explain the second step of SZ deactivation. Our study demonstrates that the monoenic allylic species (absorbing at 295 nm) detected on the catalyst during reaction, are only spectators, and not responsible for the deactivation phenomena, as reported in the literature. These species were not detected in the UV-vis spectra of MnSZ in spite of its severe deactivation. However, two bands were observed in the visible range of MnSZ spectra. An erroneous assignment of these bands in the literature could be corrected with the help of calculations. The absorption band at 580 nm arises from Mn4+ and the one at 680 nm from Mn3+
Katalysatoren auf der Basis von sulfatiertem Zirkondioxid (SZ) desaktivieren, während der Isomeriesierung von kurzkettigen Alkanen, sehr schnell. Dieser schnellen Desaktivierung zu Reaktionsbeginn folgt eine zweite Phase mit verlangsamter Desaktivierung. Das Desaktivierungverhalten während der n-Butan-Isomerisierung über sulfatiertem Zirkonoxid (SZ) und mangandotiertem sulfatierten Zirkonoxid (MnSZ; 0,5 or 2,0 wt% Mn) wurden mit Hilfe der in situ UV-vis-NIR-Spektroskopie in diffuser Reflexion, d. H. Simultane Analyse des Reaktiongases durch gaschromatographische Verfahren, untersucht. Der derzeit akzeptierte Mechanismus für den Reaktionsstart ist eine oxidative Dehydrogenierung (ODH). SZ ist ein saurer Katalysator mit wenigen aktiven Oberflächenzentren, welche leicht durch Adsorption von Edukten/Reaktionsprodukten blockiert werden können. Diese Arbeit zeigt die Dualität des Einflusses von Wasser und Buten, der Reaktionsprodukte der ODH, ihren positiven und negativen Effekt auf die n- Butan-Isomerisierung. Ein optimaler Hydratisierungsgrad des SZ ist entscheidend für die Aktivität des Katalysators. Der Hydratisierungsgrad wird maßgeblich durch die Aktivierungsbedingungen (Gaszusammensetzung und Temperatur) bestimmt. Bei nicht Einhaltung des optimalen Hydratisierungsgrades kann Wasser (Absorptionband bei 1910 nm) die aktiven Zentren blockieren und somit die Aktivität des Katalysators herabsetzen. In der vorliegenden Arbeit kann gezeigt werden, dass die Bildung von Wasser in den ersten Stunden der Reaktion für die schnelle Desaktivierung zu Reaktionsbeginn verantwortlich ist. Wie in der Literatur bereits beschrieben ist, wird Buten durch Protonierung an sauren Brønsted-Zentren, über Carbenium-Intermediate, in das entsprechende Alkoxid überführt. Die Skelettisomerisierung von n-Butan folgt einem bimolekularen Mechanismus und verläuft über die Alkylierung von Gasphasenolefinen mit sec-Butyl-Carbeniumionen. Die Gegenwart von größeren Mengen an Olefinen ist jedoch ein weiterer Grund für die Katalysatordeaktivierung. Die Zugabe von Propen zum Reaktionsgemisch führte zur Bildung von Polymeren. Die Zugabe von Sauerstoff zum Reaktionsgemisch führt zur Verbrennung von kohlenstoffhaltigen Ablagerungen auf der Katalysatoroberfläche und zur Bildung von Wasser, Kohlendioxid sowie Wärme, was vergleichbare Desaktivierungserscheinungen hervorruft. Die Freisetzung von Wärme fördert zusätzlich die Bildung von Polymeren. Oligomere konnten durch ihre Absorptionsbanden bei 370 und 450 nm auf der Katalysatoroberfläche nachgewiesen werden. Die vorliegende Arbeit zeigt, dass die Bildung oligomerer Spezies eine mögliche Ursache für den zweiten, langsameren Desaktivierungsvorgang ist. Die während der Reaktion gebildete monomere Allyl-Spezies (Absorption bei 295 nm) ist entgegen der Literatur nicht für die Desaktivierungsphänomene verantwortlich. Keine der auf SZ gefundenen allylischen Spezies konnte auf MnSZ nachgewiesen werden. Jedoch desaktiviert MnSZ sehr schnell. Zwei Banden im UV-vis Bereich von Spektren der MnSZ-Katalysatoren werden beobachtet. Die fehlerhafte Zuordnung dieser Banden in der Literatur konnte durch Simulation der Spektren korrigiert werden. Die Absorption bei 580 nm ist auf Mn4+, die bei 680 nm auf Mn3+ zurückzuführen

Actuellement, la stratégie de la production d’énergie repose sur les 3 concepts d’économie, de régénération et d’écologie. La production de biodiesel s’insère dans cette thématique et fait objet de ce travail. Un suivi cinétique de nucléation-croissance est réalisé sur des nanoparticules monodisperses d’oxo-alcoxydes de zirconium (ZOA). Ces nanoparticules sont préparées par voie sol-gel dans un réacteur à T-micro-mélangeur avec deux flux turbulents de ZNP et c dans 1-propanol à 20°C. Les nanodépôts des nanoparticules de ZOA ont été réalisés sur des substrats en silice et comparés aux nanopoudres récupérées après l’induction du sol de ZOA. Les nanodépôts et les poudres subissent un séchage à 80°C puis une imprégnation humide dans une solution aqueuse de 0,25 mol.L⁻¹ de H₂SO₄. Nous obtenons ainsi les nanodépôts catalytiques après une calcination à des températures comprises entre 500 et 700°C sous O₂. Les techniques de BET, ATG-ATD, MET, DRIFT, analyse élémentaire et DRX sont déployées pour caractériser ces catalyseurs. Les cinétiques du processus d’estérification et de transestérification ont été étudiées en fonction des conditions de la préparartion des catalyseurs nanostructurés. Les nanodépôts catalytiques acides de ZrO₂-SO₄²⁻ possèdent une activité catalytique 50 fois plus élevée que celle des nanopoudres dans la réaction d’estérification de l’acide palmitique dans le méthanol à 65°C. Les nanodépôts calcinés à 580°C ont la meilleure stabilité vis à vis des essais de recyclage. L’activité catalytique des nanodépôts est aussi valable avec d’autres charges dont la composition est similaire à celle des huiles non-comestibles puis celle des déchets gras
In this work, we have realized novel nanoparticulate catalysts ZrO₂-SO₄²⁻ for biofuel production. We have studied nucleation-growth kinetics of zirconium-oxo-alkoxy (ZOA) nanoparticles in the sol-gel process. The monodispersed nanoparticles of 3.6 nm diameter were realised in a sol-gel reactor with rapid (turbulent) micro-mixing of liquid solutions containing ZNP and H₂O in 1-propanol at 20°C. The nanocoatings were realised of stable colloids of ZOA nanoparticles on silica beads along with common powders obtained after precipitation of unstable colloids. The acid ZrO₂-SO₄²⁻" catalysts were prepared after drying at 80°C, wet impregnation in 0.25 mol.L⁻¹ aqueous solution of sulfuric acid and subsequent thermal treatment between 500 and 700°C and studied with BET, DTA-DSC, TEM, DRIFT, elemental analysis, DRX and other methods. The catalyst nanocoatings calcinated at 580°C showed strong activity in esterification reaction of palmitic acid in methanol at 65°C, which is about 50 times higher than that of nanopowders, and also possesses the highest stability towards recycling. Tha catalytic performance of catalytic nanocoatings was also confirmed on unedible and waste oils

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
O cÃncer à uma doenÃa que afeta cada vez mais um nÃmero maior de pessoas em todo o mundo. As terapias atuais utilizadas para o tratamento do cÃncer sÃo ainda insatisfatÃrias. Produtos naturais tÃm sido avaliados para seleÃÃo de compostos ativos, capazes de reduzir os tumores malignos de uma forma mais eficiente e com menos efeitos indesejÃveis. O polissacarÃdeo sulfatado extraÃdo da alga Champia feldmannii (Cf-PLS) foi testado para avaliaÃÃo do seu potencial antitumoral e imunoestimulante. AlÃm disso, foram feitos testes de toxicidade do fÃgado, rins e baÃo apÃs o tratamento com Cf-PLS. Os resultados demonstraram que Cf-PLS nÃo apresenta citotoxicidade in vitro, mas à capaz de reduzir o tumor Sarcoma 180 implantado em camundongos em 48,16% e 48,62% nas doses de 10 e 25mg/kg, respectivamente, e reduz 68,32% quando adiministrado juntamente com 5-Fluorouracil (5-FU). As anÃlises do fÃgado e rins revelaram que houve certa toxicidade no rim, com Ãreas de necrose tubular aguda na maior dose. Os testes da perfusÃo renal revelaram que Cf-PLS causa aumento da pressÃo de perfusÃo, resistÃncia vascular renal, ritmo de filtraÃÃo glomerular e fluxo urinÃrio, alÃm da excreÃÃo de sÃdio, cloreto e potÃssio. NÃo houve alteraÃÃes nos percentuais de transporte totais ou tubular proximais de sÃdio, cloreto ou potÃssio. Houve discreta deposiÃÃo protÃica nos glomÃrulos e tÃbulos renais. NÃo houve alteraÃÃes nas anÃlises bioquÃmicas e os testes hematolÃgicos revelaram uma leucopenia causada por 5-FU, entretanto esta foi revertida pelo tratamento com Cf-PLS. TambÃm foi demonstrado que Cf-PLS age como agente imunoestimulante e imunomodulador, aumentando a produÃÃo de anticorpos totais e especÃficos contra Cf-PLS e OVA, aumentando tambÃm a polpa branca e o nÃmero de megacariÃcitos nos baÃos dos animais tratados e induzindo a migraÃÃo de neutrÃfilos para a cavidade peritoneal de camundongos. Pode-se concluir que Cf-PLS apresenta atividade antitumoral e que isso pode estar relacionado com suas propriedades imunoestimulantes.
Cancer is a desease that occurs in a larger number of people each year. The therapies used to treat it are still not satisfatory. Natural products have been studied to select new compounds capable of reducing tumours and with fewer side effects. The sulfated polysaccharide isolated from the seaweed C. feldmannii (Cf-PLS) was investigated for its antitumor and immunostimulating properties and also for the toxicological aspects related to Cf-PLS treatment. The Cf-PLS did not show any significant in vitro cytotoxic effect, but showed a strong in vivo antitumor effect. The inhibition rates of sarcoma 180 tumor development were 48.16 % and 48.62% at the doses of 10 and 25 mg/kg, respectively, and 68.32% when treated together with 5-fluorouracil (5-FU). The histopathological analysis of liver and kidney showed that the kidneys were affected by Cf-PLS-treatment, presenting some focal areas of acute tubular necrosis at the higher dose. Cf-PLS caused considerable changes in renal physiology, as shown by an increase in parameters such as perfusion pressure, renal vascular resistance, glomerular filtration rate, urinary flow and sodium, chloride and potassium excretion. Neither enzymatic activity of alanine aminotransferase, urea nor creatinin levels were significantly altered. In hematological analyses, leucopeny was observed after 5-FU treatment, but this effect was prevented when the treatment was associated with the Cf-PLS. It was also demonstrated that Cf-PLS acts as an immunomodulatory agent, raising the production of specific antibodies, and also increasing the production of OVA-specific antibodies. In addition, it induced a discreet increase of the white pulp and nest of megakaryocytic in spleen of treated mice and the neutrophil migration to the intraperitoneal cavity of mice. In conclusion, Cf-PLS has some interesting antitumour activity that could be associated with its immunostimulanting properties.

Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

Sulfated low molecular weight lignins (LMWLs), CDSO3 and FDSO3, designed recently as macromolecular mimetics of heparin, were found to exhibit potent anticoagulant activity. Small molecules based on the same scaffold, SBD and SBT, showed promising thrombin inhibition. We were able to address the mechanism of the inhibition using Michaelis-Menten kinetics. All the molecules were found to be allosterically impairing thrombin activity using either noncompetitive or uncompetitive mechanism. Absence of competition with hirugen, an exosite 1 ligand, and competition with polymeric heparin points to exosite 2 as the site of interaction for these inhibitors. Yet mixed competition results with other exosite 2 ligands indicated that the molecules utilize different sub-sites within exosite 2 for interaction. Site-directed mutagenesis was used to pin point the key residues important for inhibition. All of all positively charged exosite 2 residues were mutated one at a time to alanine to abolish its charge. The data showed that Arg93 and Arg175 are the major residues involved in CDSO3 binding. FDSO3 showed a progressively greater defect in inhibition with double point mutations, the triple mutant Arg93,97,101Ala displayed a 50 fold drop in inhibition. A single mutant, Arg173Ala, displayed 22-fold reduction in IC50 of SBD, while Arg233Ala was the only mutation that impaired SBT inhibition. This proves the fact that inspite of the structural similarity between the two polymers and the two small molecules, thtey do not share the same binding space in exosite 2. To understand the types of interactions involved in thrombin interaction with the polymers, we resorted to salt-dependence studies. This showed that CDSO3 had fewer ionic contacts with thrombin, with most of its binding energy derived from non-ionic interactions. FDSO3 on the other hand had a balanced contribution of ionic and non-ionic forces. Thermodynamic studies showed that both polymers have a positive ΔCp of binding, which proves the involvement of electrostatic forces and signals the burial of the polar residues on thrombin exosite 2. These molecules offer a rare chance to study thrombin allostery. Little is known about the interplay between exosite 2, active site and sodium binding site. The allosteric nature of inhibition indicated that, for the first time, a link is proven to exist between exosite 2 and the active site that could be used to inhibit the enzyme. The presence of sodium was found to enhance the binding of FDSO3 at exosite 2, which establish the energetic coupling between exosite 2 and sodium binding site. The results identify novel binding sub-sites within exosite 2 that are energetically coupled to thrombin’s catalytic function and linked to the sodium binding site. The design of high affinity small molecules based on LMWLs scaffold presents major opportunities for developing clinically relevant, allosteric modulators of thrombin.

Two novel, single-isomer, sulfated cyclodextrins, the sodium salts of heptakis(2- O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose (HMAS) and heptakis(2-O-methyl- 6-O-sulfo)cyclomaltoheptaose (HMS) were used as chiral resolving agents in both aqueous and non-aqueous electrophoretic separation of a set of pharmaceutically active weak base enantiomers. Enantiomers of twenty one of the twenty four weak bases were baseline resolved in one or more of the background electrolytes (BGE’s) used. An eight-step synthetic method was used to produce, on a large scale, the title compounds in greater than 97% purity. The purity of the synthetic intermediates and the final products were characterized by HPLC-ELSD and indirect UV-detection capillary electrophoresis (CE), respectively. X-ray crystallography, MALDI-TOF mass spectrometry and 1H as well as 13C NMR spectroscopy allowed for unambiguous characterization of the structure of each intermediate and the final product.

Les glycosaminoglycannes (GAGs) sont des molécules polysaccharidiques polysulfatées intervenant dans des processus aussi variés que la prolifération, différenciation ou migration cellulaire, la coagulation sanguine ou l‟infection virale. Il est généralement admis qu‟une séquence particulière de GAG doit être associée à une fonction biologique spécifique. Les structures chimiques globales des GAGs sont connues. Cependant, contrairement au séquençage des gènes ou des protéines, la détermination de la séquence saccharidique exacte impliquée dans une fonction biologique particulière n‟est encore pas possible. Le séquençage « glycomique » constitue donc un enjeu majeur. L‟une des technologies les plus novatrices pour aborder ce problème de séquençage des GAGs semble être l‟impression moléculaire. En effet, elle permet d‟obtenir des polymères (MIPs pour Molecular Imprinted Polymer) spécifiquement imprimés par la forme structurale d‟une molécule cible.En nous appuyant sur des travaux antérieurs réalisés avec des modèles saccharidiques sulfatés simples, nous avons appliqué cette technologie à la reconnaissance de glycannes sulfatés complexes d‟intérêt biologique tels qu‟une héparine de bas poids moléculaire ou un mimétique ayant une activité anticoagulante. Il a été démontré une reconnaissance spécifique et sélective selon la molécule étudiée à l‟aide de MIPs spécialement conçus pour chaque GAG. De plus, nous avons obtenu des MIPs qui, en immobilisant temporairement un sucre, permettraient leur substitution de façon stéréospécifique. La détermination des conditions optimales de synthèse des MIPs s‟est avéré une étape nécessaire à l‟obtention d‟une bonne reconnaissance. Ces travaux ouvrent des perspectives d‟application de la technique d‟impression moléculaire à l‟analyse des séquences de GAGs d‟intérêt biologique
Glycosaminoglycans (GAGs) are polysulfated polysaccharide molecules involved in many biological processes such as cellular proliferation, differentiation or migration, blood clotting or viral infection. It is generally admitted that a particular GAG sequence is connected to a specific biological function. Depending on their composition in disaccharides, GAGs are classified into subfamilies whose overall chemical structures are known. Unlike gene or protein sequencing, determination of the exact saccharidic sequence involved in a particular biological function is not yet possible with the available technological tools. "Glycomics" is a real challenge nowadays. One of the most innovative technologies to achieve this goal seems to be the molecular imprinting. Indeed, it provides polymers (MIPs for Molecular Imprinted Polymer) imprinted by the structural form of a target molecule.Based on previous studies performed with simple sulfated saccharides, this technology has been applied to the recognition of complex sulfated glycans. MIPs were achieved demonstrating specific and selective recognition for a Low Molecular Weight Heparin or a synthetic anticoagulant mimetic. Other MIPs were able to temporally immobilize sugars which make them available for stereo-specific modifications. Screening of optimal synthesis conditions of MIPs appeared a necessary step to obtain a specific and selective recognition. These studies open further possibilities to analyze GAG sequences carrying biological functions by the molecular imprinting technology

Sulfated glycoprotein-2 (SGP-2) is an acidic glycoprotein that has recently been discovered in numerous tissues including brain. This glycoprotein is involved in the response to diverse types of neuronal injury and synaptic remodelling. It is well established that estradiol treatment results in selective destruction of $ beta$-endorphin neurons in the hypothalamic arcuate nucleus. The question addressed by this thesis is what effect SGP-2 plays in this estradiol-induced pathology. We have used immunocytochemistry to elucidate the distribution of SGP-2 in the normal and estradiol-lesioned hypothalamus. Unexpectedly, SGP-2-immunopositive perikarya occurred in a highly specific distribution in the normal rat hypothalamus. SGP-2-reactive neurons were localized to the medial preoptic area (MPOA), supraoptic nucleus (SON), paraventricular nucleus (PVN), dorsomedial nucleus (DMN) and perifornical area (PA) of the hypothalamus. Both the male and female hypothalamus exhibited similar distributions. The neuropil as well as small cell profiles consistent with glia appeared free of SGP-2 labelling. Estradiol-induced loss of $ beta$-endorphin resulted in an increase in the number of SGP-2-reactive neurons only within the MPOA. Small cell profiles appeared labelled, after estradiol-valerate (EV) treatment, mainly within the MPOA and immunopositive deposits were associated with the neuropil of the PVN, PA and MPOA. Since, vitamin E, which blocks the neurotoxic effect of estradiol also prevents the EV-induced changes in SGP-2 distribution, the increase of SGP-2-immunopositive cells and presence of neuropil deposits result from the deafferentation engendered by the estradiol-induced lesion rather than a direct effect of estradiol. The exact role SGP-2 plays in the hypothalamus is still unknown, however we suggest SGP-2 acts as a mediator of synaptogenesis in both the normal and pathological states. We conclude that the SGP-2 response in the MPOA is possibly related to the selecti

nÃo hÃ
PolissacarÃdeos sulfatados de algas marinhas sÃo polÃmeros heterogÃneos que representam biomolÃculas de interesse comercial, principalmente nas indÃstrias alimentÃcia e farmacÃutica. O presente trabalho teve como finalidade avaliar os efeitos dos polissacarÃdeos sulfatados totais (PST) da alga marinha verde Caulerpa mexicana em modelos clÃssicos de nocicepÃÃo e inflamaÃÃo aguda em animais. Foram utilizados camundongos Swiss machos (18-25 g) e ratos Wistar machos (120-260 g). O rendimento de PST obtidos da extraÃÃo por digestÃo enzimÃtica foi de 1,7%. Na anÃlise por espectroscopia na regiÃo do infravermelho foram caracterizados como polissacarÃdeos contendo, principalmente, galactose sulfatada e Ãcido urÃnico. Os PST da alga C. mexicana foram capazes de reduzir (p<0,05) o nÃmero de contorÃÃes abdominais induzidas por Ãcido acÃtico (1%) em 31,8; 74,5 e 88,9% nas doses de 5, 10 e 20 (mg/kg; i.v.), respectivamente. No teste da formalina, os PST inibiram (p<0,05) o tempo de lambedura da pata somente na segunda fase do teste (88,6 e 98,5% para as doses 10 e 20 mg/kg; i.v., respectivamente). No teste da placa quente, os PST nÃo demonstraram efeito antinociceptivo no decorrer dos 90 minutos do teste. Desta forma, os resultados sugerem que os PST exerceram um efeito antinociceptivo de aÃÃo perifÃrica. Em adiÃÃo, os PST (5, 10 e 20 mg/kg) apresentaram efeito anti-inflamatÃrio quando administrados por via s.c. em ratos Wistar no modelo de edema de pata induzido por carragenana, dextrana e histamina. Para analisar o envolvimento da via heme oxigenase (HO) no efeito anti-inflamatÃrio dos PST (20 mg/kg; s.c.), os animais foram prÃ-tratados por via s.c. com um inibidor de HO especÃfico (zinco protopofirina IX). Os resultados obtidos demonstraram que os PST foram capazes de reduzir o edema de pata induzido por carragenana na terceira hora, cujo resultado foi comprovado pela quantificaÃÃo da mieloperoxidase. AlÃm disso, os PST reduziram o edema induzido por dextrana ou histamina em modelos de edema de pata nos primeiros 30 minutos apÃs a aplicaÃÃo dos agentes flogÃsticos. O efeito anti-inflamatÃrio dos PST no edema de pata induzido por carragenana nÃo foi observado apÃs inibiÃÃo prÃvia por zinco protopofirina IX. Portanto, sugerimos que o efeito anti-inflamatÃrio dos PST da alga C. mexicana pode estar relacionado com a inibiÃÃo da liberaÃÃo de histamina, reduÃÃo da migraÃÃo celular e ativaÃÃo da via da HO. Para avaliar os efeitos sistÃmicos dos PST, estes foram administrados por via i.v. (20 mg/kg) em camundongos em dose Ãnica e as alteraÃÃes sistÃmicas avaliadas durante 48 h. Os resultados obtidos demonstraram que os PST nÃo causaram mortalidade e nem alteraÃÃes macroscÃpicas significativas dos ÃrgÃos e dos parÃmetros bioquÃmicos avaliados, sendo, portanto, considerados seguros na dose testada. Em resumo, os PST da alga C. mexicana apresentaram efeitos antinociceptivos e anti-inflamatÃrios significativos, tornando-se uma importante ferramenta biotecnolÃgica para estudos posteriores.
Sulfated polysaccharides from seaweed are heterogeneous polymers representing biomolecules of commercial interest, especially in food and pharmaceutical industries. The aim this study was to evaluate the effects of total sulfated polysaccharides (TSP) of the green seaweed Caulerpa mexicana in classical models of nociception and acute inflammation in animals. Swiss male mice (18-25 g) and male Wistar rats (120-260 g) were used. The yield of the extraction of TSP obtained by enzymatic digestion was 1.7%. In analysis by Fourier transformed infrared were characterized as polysaccharides containing mainly sulfated galactose and uronic acid. The TSP of the alga C. mexicana were able to decrease (p <0.05) the number of writhes induced by acetic acid (1%) on 31.8, 74.5 and 88.9% at doses of 5, 10 and 20 mg/kg; i.v., respectively. In the formalin test, the TSP inhibited (p <0.05) the licking time of the paw only on the second of the test phase (88.6 and 98.5% for the doses 10 and 20 mg/kg; i.v., respectively). In the hot plate test, the TSP did not show antinociceptive effect in the course of 90 minutes of the test. Thus, the results suggest that the TSP presented analgesic effect by a peripheral action. In addition, TSP (5, 10 and 20 mg/kg) showed anti-inflammatory effect when administered s.c. in rats on paw edema models induced by carrageenan, dextran or histamine. To analyze the involvement of heme oxygenase (HO) pathway in the anti-inflammatory effect of TSP (20 mg/kg, sc), the animals were pretreated (s.c.) with a specific HO inhibitor (zinc protoporphirin IX). The results showed that the TSP were able to reduce the paw edema induced by carrageenan in the third hour, which was confirmed by myeloperoxidase quantification. In addition, the TSP reduced the edema induced by dextran or histamine in paw edema models in the first thirty minutes after application the flogistic agents. The anti-inflammatory effect of TSP on paw edema induced by carrageenan was not observed after prior inhibition by zinc protoporphyrin IX. Therefore, we suggest that the anti-inflammatory effect of TSP of the alga C. mexicana algae may be related to inhibition of the histamine release, reduction of cell migration and activation of the HO pathway. To evaluate the systemic effects of TSP, they were administered (i.v.) in mice (20 mg/kg) in a single-dose and evaluated the systemic changes for 48 h. The results showed that the TSP did not cause mortality or significant alterations on organs and on biochemical parameters being considered safe in the tested dose. In summary, the TSP from the algae C. mexicana have showed significant antinociceptive and anti-inflammatory effects, becoming an important biotechnological tool for further studies.

The alternative splicing of the prosaposin gene results in the inclusion or exclusion of a 9 bp insertion of exon 8, which is located in the saposin B domain of this gene. Thus, exon 8 codes for three amino acid residues (Gin-Asp-Gln), which may potentially be present or absent in human prosaposin. Recently, it was shown that the alternative splicing of the prosaposin gene may be tissue specific and that a possible function of the three amino acid insertion could be to alter the binding specificity of saposin B towards different glycosphingolipids. We have recently cloned the murine SGP-1 gene and found that it also contains the 9 bp exon 8. In the present study we have used reverse transcription-polymerase chain reaction (RT-PCR) to study the distribution of the alternatively spliced mRNAs in several tissues. We have also used Northern Blot analysis to confirm the expression and stability of these transcripts, and light microscope immunocytochemistry to examine whether or not alternative splicing affects the translation of the mRNA transcripts into mature proteins. (Abstract shortened by UMI.)

Moltes activitats industrials generen emissions que contenen compostos de sofre tant en efluents líquids com emissions gasoses, que majoritàriament són tractades mitjançant processos fisicoquímics. El sulfat es troba generalment a les aigües residuals d’aquestes indústries, com la indústria paperera, la farmacèutica, la minera o l’alimentària. Com a tal, el sulfat no és un compost nociu, però si s’aboca als rius o sistemes de clavegueram, pot generar-se un desequilibri del cicle del sofre. Dins d’aquest cicle, el producte final de la reducció de compostos és el sulfur d’hidrogen (H2S). Aquest compost és corrosiu, olorós i s’ha demostrat que és tòxic en baixes concentracions. Per aquests motius, és necessari desenvolupar alternatives respectuoses amb el medi ambient per a tractar i valoritzar no només les emissions de SO2 sinó també els efluents líquids rics en sofre. A més, podria recuperar-se sofre elemental d’aquests efluents, la qual cosa brindaria l’oportunitat de recuperar recursos en el marc de l’economia circular. Amb aquestes premisses, el projecte SONOVA, en el qual s’emmarca aquesta tesi, va desenvolupar un procés integral de tractament del SOx i el NOx provinent de gasos de combustió mitjançant processos biològics, econòmics, robustos i respectuosos amb el medi ambient que també tenen en compte la reutilització d’energia i recursos al llarg del procés, així com la valorització de residus. El procés proposat es basa en una primera doble etapa per a l’absorció selectiva de SOx i NOx; una segona etapa biològica per a reduir el sulfat de la primera etapa d’absorció a sulfur d’hidrogen (que és l’objectiu d’estudi d’aquesta tesi); i una tercera etapa biològica per a l’oxidació del sulfur d’hidrogen a sofre elemental i la seva posterior recuperació. El desenvolupament de sistemes biològics, com el reactor de llit anaerobi amb flux ascendent (UASB), han estat implementats per al tractament de diverses aigües residuals i per a la digestió anaeròbia. En aquesta tesi, s’ha estudiat l’ús d’aquest tipus de reactor UASB pel tractament d’aigües sintètiques amb sulfat, específicament, es va seleccionar el glicerol cru com a font de carboni i donador d’electrons. Es van utilitzar tant processos fisicoquímics com tècniques de biologia molecular per a obtenir un major coneixement del procés. Es va estudiar la influència de possibles inhibicions i la competència entre els bacteris reductors de sulfat i els metanògens a fi de millorar l’eliminació de sulfat i la producció de sulfur. Es va observar que en les operacions a llarg termini (després de 200 dies aproximadament) els metanògens desapareixen del sistema i els bacteris reductors del sulfat són els que colonitzen. No obstant això, es va observar una acumulació d’acetat a conseqüència de la desaparició dels metanògens, la qual cosa va donar lloc a una pèrdua de la font de carboni a la sortida del reactor que podria haver-se utilitzat per a produir sulfur. Les operacions a llarg termini permeten detectar altres limitacions del sistema. Al llarg de les operacions en UASB dutes a terme en aquesta tesi, es va observar una pèrdua de l’estructura granular i el creixement d’una biopel·lícula no metanogènica ni sulfat reductora no identificada. Aquesta biopel·lícula, anomenada slime al llarg d’aquesta tesi, es va considerar com un factor crucial que afectava el sistema biològic, conferint propietats com la viscositat al llit granular. En conseqüència, es van poder observar problemes relacionats amb la limitació de transferència de matèria, que afectava també l’activitat sulfat reductora dels grànuls i que va conduir a operacions fallides.
Muchas actividades industriales generan emisiones que contienen compuestos de azufre tanto en efluentes líquidos como emisiones gaseosas, que mayoritariamente son tratadas mediante procesos fisicoquímicos. El sulfato se encuentra generalmente en las aguas residuales de estas industrias, como la industria papelera, la farmacéutica, la minera o la alimentaria. Como tal, el sulfato no es un compuesto nocivo, pero si se vierte en los ríos o en los sistemas de alcantarillado, puede generarse un desequilibrio en el ciclo del azufre. Dentro de este ciclo, el producto final de la reducción de compuestos dentro del mismo es el sulfuro de hidrógeno (H2S). Este compuesto es corrosivo, oloroso y se ha demostrado que es tóxico en bajas concentraciones. Por estos motivos, es necesario desarrollar alternativas respetuosas con el medio ambiente para tratar y valorizar no sólo las emisiones de SO2 sino también los efluentes líquidos ricos en azufre. Además, podría recuperarse azufre elemental de esos efluentes, lo que brindaría la oportunidad de recuperar recursos en el marco de la economía circular. Con estas premisas, el proyecto SONOVA, en el cual se enmarca esta tesis, desarrolló un proceso integral de tratamiento del SOx y el NOx proveniente de gases de combustión mediante procesos biológicos, económicos, robustos y respetuosos con el medio ambiente que también tienen en cuenta la reutilización de energía y recursos a lo largo del proceso, así como la valorización de residuos. El proceso propuesto se basa en una primera doble etapa para la absorción selectiva de SOx y NOx; una segunda etapa biológica para reducir el sulfato de la primera etapa de absorción a sulfuro de hidrógeno (que es el objetivo de estudio de esta tesis); y una tercera etapa biológica para la oxidación del sulfuro de hidrógeno a azufre elemental y su posterior recuperación. El desarrollado de sistemas, como el reactor de lecho de lodo anaerobio de flujo ascendente (UASB), han sido implementados para el tratamiento de diversas aguas residuales y para la digestión anaerobia. En esta tesis, se estudió el uso de este tipo de reactor UASB para el tratamiento de aguas sintéticas con sulfato, específicamente, se seleccionó el glicerol crudo como fuente de carbono y donador de electrones. Se utilizaron tanto procesos fisicoquímicos como técnicas de biología molecular para obtener un mayor conocimiento del proceso. Se estudió la influencia de posibles inhibiciones y la competencia entre las bacterias sulfato reductoras y los metanógenos a fin de mejorar la eliminación de sulfato y la producción de sulfuro. Se observó que en las operaciones a largo plazo (después de 200 días aproximadamente) los metanógenos desaparecen del sistema y las bacterias sulfato reductoras son las que lo colonizan. Sin embargo, se observó una acumulación de acetato como consecuencia de la desaparición de los metanógenos, lo que dio lugar a una pérdida de la fuente de carbono en la salida del reactor que podría haberse utilizado para producir sulfuro. Las operaciones a largo plazo permiten detectar otras limitaciones del sistema. A lo largo de las operaciones del UASB llevadas a cabo en esta tesis, se observó una pérdida de la estructura granular y el crecimiento de una biopelícula no metanogénica ni sulfatoreductora no identificada. Esta biopelícula, llamada slime a lo largo de esta tesis, se consideró como un factor crucial que afectaba a nuestro sistema, confiriendo propiedades como la viscosidad al lodo granular. En consecuencia, se pudieron observar problemas relacionados con la limitación de transferencia de materia, que afectaba también a la actividad sulfato reductora de los gránulos y que condujo a operaciones fallidas.
Many industrial activities generate effluents containing sulfur compounds, both as liquid or gaseous emissions, which are mainly treated through physical-chemical processes. Sulfate is generally present in wastewaters coming from paper, pharmaceutical, mining or food processing industries, among others. As such, sulfate is not a harmful compound, but if it is poured into rivers or sewage systems, an imbalance in the overall sulfur cycle can be generated. Inside this cycle, the last product after the reduction of sulfur compounds is hydrogen sulfide (H2S). This compound is corrosive, odorous and toxic at low concentrations. For these reasons, there is a need to develop environmentally friendly alternatives to valorize not only gaseous emissions, such as SO2 emissions, but also S-rich liquid effluents. In addition, a further recovery of elemental sulfur from these effluents could be obtained providing an opportunity to recover resources in the framework of the circular economy. With these premises, the SONOVA project, in which this thesis is enclosed, is based in the development of a comprehensive treatment process to valorize SOx and NOx from flue gases by economical, robust and environmentally friendly biological methods. It also takes into account the reuse of energy and resources along the process as well as residues valorization. The proposed process is based on a first double stage for selective absorption of SOx and NOx; a second biological step for reducing the sulfate from the first absorption stage to hydrogen sulfide (which is the focus of this thesis); and a third biological stage for the oxidation of hydrogen sulfide to elemental sulfur and its subsequent recovery. Biological-based systems, such as Up-flow Anaerobic Sludge Bed (UASB) reactors, have been developed and implemented world-wide to treat many types of wastewater and to produce biogas through anaerobic digestion. In this thesis, the use of an UASB reactor for the treatment of synthetic wastewater with sulfate was studied, specifically selecting crude glycerol as carbon source and electron donor. Both physical-chemical processes and molecular biology techniques were used to get a broad knowledge of the anaerobic process. The influence of possible inhibitions and competition between sulfate reducers and methanogens was studied in order to improve sulfate removal and sulfide production. It was observed that in long-term operations (after 200 days approximately) methanogens were washed out from the system and sulfate reducers colonized the reactor sludge. However, acetate accumulation was observed because of the disappearance of methanogens, leading to a loss of carbon source in the outlet of the reactor that could have been used to produce sulfide in the UASB. Long-term performances allow detecting further limitations of the system. A loss of granular structure and the growth of unidentified non-sulfate reducer, non-methanogenic biofilm was observed during UASB operations along this thesis. This biofilm, called slime substance along this thesis, was found to be a crucial factor affecting our system, conferring properties such as viscosity to the sludge. Consequently, problems related to mass transfer limitations could be observed, affecting as well, the sulfate reducing activity of the granules and leading to failure operations. Finally, since the accumulation of acetate could not be avoided, experiments were designed to pursue the enrichment of acetate degrading sulfate reducing bacteria in serum bottles, with the final objective of improving sulfidogenesis. In addition, isolation of potential acetate-utilizing sulfate reducers was also pursued. Unfortunately, a culture able to perform sulfate reduction with acetate was not developed during the enrichment experiments. Therefore, further research is needed to enhance the operation in terms of organic matter consumption and sulfide productivity in the long-term.

Biodiesel is currently produced by homogeneous catalysis. More recently however, heterogeneous catalysis is being considered as a cheaper alternative to the homogeneous process. In this research project, heterogeneous catalysts of zirconium oxide were produced by impregnation. Zirconium oxide impregnation with sulfuric acid produced acidic solid catalysts. It was determined that impregnation and calcination at 550^oC (SO₄/ZrO₂-550^oC) produced the best catalyst for palmitic acid esterification with 10 wt % as the optimum concentration in esterification of palmitic acid. SO₄/ZrO₂-550^oC was successfully recycled for eight consecutive runs before permanent deactivation. Its sulfur content was 1.04 wt % using SEM-EDS and 2.05 wt % using XPS for characterization. BET surface area was 90.89 m2/g. The reaction mechanism over Brønsted acid (SO₄/ZrO₂-550^oC) and Lewis acid (Al₂O₃) catalysts obeyed Eley-Rideal kinetics with palmitic acid and methanol adsorbed on the active site respectively. Zirconium oxide was also impregnated with sodium hydroxide to produce basic catalysts. The best catalyst was produced when zirconium oxide was impregnated with 1.5 M NaOH and calcined at 600^oC. Soybean oil was completely converted to biodiesel with 10 wt % catalyst and 1:6 oil to methanol. A mixture of the base catalyst with 30 wt % SO₄/ZrO₂-550^oC effectively converted soybean oil containing 5% oleic acid indicating that this mixture could be used for waste oils. The reaction was first order with respect to triglyceride and second order with respect to methanol. The activation energy was 49.35 kJ/mol and the reaction mechanism obeyed Langmuir-Hinshelwood kinetics.
Master of Science

Cystoseira compressa et Jania adhaerens sont deux macro-algues marines de la mer méditerranée bien répandues le long des côtes Tunisiennes et non exploitées. Il ressort de cette étude que ces algues pourraient être utilisées comme une source d’ingrédients fonctionnels d’origine naturelle pour la production de burgers à base de chair de poissons d’eau douce (Barbus barbus). De plus, ces travaux ont conduit à l’obtention d’un fucoïdane (CCF) et un alginate de sodium (CCSA) comme polysaccharides matriciels de C. compressa. CCF (1,05 × 105 g/mol) est un hétérogalactofucane sulfaté (14,65 %) composé d’une chaîne principale de α-(1,3 ; 1,4)-l-Fucp ramifiée (31,84 %) en positions O-4 et O-3 par des monosaccharides terminaux et des chaînes latérales. CCSA (Mw = 1 × 105 g/mol) est composé de 56 % de α-l-GulA (G) et 44 % de β-d-ManA (M) (M/G = 0,77). Ce polyuronide est constitué de 93 % d’homoblocs (FGG = 53 % et FMM = 40 %) et de 6 % d’hétéroblocs (FMG = 3 % et FGM = 3 %). Les analyses rhéologiques et biologiques ont montré que CCF et CCSA présentent un comportement rhéofluidifiant ayant des propriétés de liquide visqueux avec des propriétés antioxydantes. Le polysaccharide issu de J. adhaerens (JSP) est un hétéroxylogalactane de Mw = 8,0 × 105 g/mol et est constitué de résidus disaccharidiques répétitifs de [→3)-β-d-Galp-(1,4)-α-l-Galp-(1→)]n et [(→3)-β-d-Galp-(1,4)-3,6-α-l-AnGalp-(1→)]n substitués principalement en position O-6 des résidus (1,3)-β-d-Galp et en positions O-2 et O-3 des résidus (1,4)-α-l-Galp par des T-β-d-Xylp, de groupements sulfate et/ou méthoxyle. Ce galactane sulfaté possède un comportement pseudoplastique typique d’un fluide viscoélastique, ayant des propriétés de gel faible
Cystoseira compressa and Jania adhaerens are two seaweeds widespread on the Tunisian coasts and were not exploited. They have been used as natural ingredients to produce new canned fish burgers prepared from minced fish of common barbel (Barbus barbus). In addition, this work led to the extraction of a fucoidan (CCF) and a sodium alginate (CCSA) as matrix polysaccharides of C. compressa. CCF (Mw=1.05 × 105 g/mol) was a sulfated (14.65%) heterogalactofucan composed of α-(1,3) and α-(1,4)-linked l-Fucp as main backbone, which could be branched (31.8%) in O-3 and O-4 positions by terminal monosaccharides and side chains. CCSA (Mw = 1 × 105 g/mol) was composed of 56% α-l-GulA (G) and 44% β-d-ManA (M) (M/G = 0.77). The CCSA linear backbone was constituted by 93% of homoblocks (FGG = 53% and FMM = 40%) and 6% of heteroblocks (FMG = 3% and FGM = 3%). Rheological and biological investigations showed that CCF and CCSA solutions exhibited shear-thinning and fluid-like viscoelastic behaviors with antioxidant properties. A sulfated xylogalactan-rich fraction (JSP) was extracted from J. adhaerens. JSP (Mw = 8.0 × 105 g/mol) was mainly constituted by the agaran disaccharidic repeating residues (→3)-β-d-Galp-(1,4)-α-l-Galp-(1→)n and (→3)-β-d-Galp-(1,4)-3,6-α-l-AnGalp-(1→)n mainly substituted on O-6 of (1,3)-β-d-Galp residues and in O-2 and O-3 positions of (1,4)-α-l-Galp residues by T-β-d-Xylp, methoxy and/or sulfate groups. JSP solutions displayed a shear-thinning behavior with a great viscoelasticity character, having weak gel properties

Sulfated carbohydrates play key roles in a wide range of biological processes such as blood clotting, viral entry into cells, amyloidogenesis, neurite outgrowth, tumor growth and metastasis. However, their synthesis still remains a considerable challenge. A general approach to the synthesis of sulfated carbohydrates was examined in which sulfate group is incorporated at the beginning of the syntheses as a protected sulfodiester. Towards this end, a series of sulfuryl imidazolium salts (SIS), a new class of sulfating agents, was prepared and examined as reagents for incorporating 2,2,2-trichloroethyl-protected sulfate esters into monosaccharides. The SIS that contained a 1,2-dimethylimidazolium moiety proved to be a superior sulfating agent compared to SIS bearing no alkyl groups or bulkier alkyl groups on the imidazolium ring. Difficult O- and N- sulfations that required prolonged reaction times and a large excess of the SIS bearing a 1-methylimidazolium group were achieved in high yield and in less time when employing less than half the 1,2-dimethylimidazolium derivative. Efforts were then made to apply the sulfate protecting group strategy to the total synthesis of a class of chondroitin sulfate glycosaminoglycans. These studies revealed some of the limitations of the sulfate protecting group approach to the synthesis of sulfated oligosaccharides. Studies on the selective introduction and isomerization of the carbobenzyloxy protecting group into 2,3-diols of 4,6-O-benzylidene galactose derivatives are also reported.

博士
國防醫學院
生命科學研究所
91
Japanese encephalitis virus (JEV) infects a broad range of cell types in vitro, though little is known about the initial events of JEV infection. In the present study, we found that highly sulfated glycosaminoglycans (GAGs) are involved in infection of both neurovirulent (RP-9) and attenuated (RP-2ms) JEV strains. Competition experiments using highly sulfated GAGs, heparin and dextran sulfate, demonstrated an inhibition of JEV’s attachment and subsequent infection of BHK-21 cells. Treatment of target cells by a potent sulfation inhibitor, sodium chlorate, greatly reduced viral binding ability as well as infection, suggesting a critical role of GAGs’ sulfation status on the cellular surface in JEV infection. This phenomenon was confirmed by the manifestation of a distinct binding efficiency of JEV to the wild-type CHO cell line and its mutants with defects in GAG biosynthesis. We also demonstrated the binding of JEV particles and virus envelope glycoprotein to immobilized heparin beads. Furthermore, the addition of heparin suppressed the cytopathic effects induced by JEV infection in cultured cells. Our results establish that the highly sulfated form of GAGs on cell surfaces plays a determining role in the early stage of in vitro JEV infection.

碩士
國立中正大學
化學暨生物化學研究所
107
Human milk oligosaccharides are structurally diverse and unconjugated glycans. They are important to the infant growth since they can increase the amount of probiotics in small intestine. The pathological functions of sulfated human milk oligosaccharides are poorly understood since they are difficult to obtain. To get the sulfated oligosaccharides for future biological study, We have synthesized glycosyl acceptor (compound 9) and glycosyl donors (compound 12, compound 13, compound 16) to prepare trisaccharide (compound 20). Followed by selectively sulfonylation and functional group transformation, the core trisaccharide structure (compound 2) of TFpLNH sulfate III (1) was achieved. The compound 2 could extended by different glycosyltransferases and the glycans could applied for the investigation of pathological functions.

Sulfated zirconia was prepared using a two-step sol-gel method. The isomerization of n-butane was used as a probe reaction. The formation of coke on sulfated zirconia during the reaction was quantified as a function of time on stream and temperature using a TGA/FTIR technique. The formation of coke was essentially linear with time on stream when the isomerization reaction was performed at 200$\sp\circ$C. When the amount of coke deposited reached 0.04 wt%, the catalytic isomerization activity was observed to decrease to 10% of its initial value. When the reaction temperature was increased, the higher selectivity to cracked products resulted in an increase in coke formation and a higher rate of deactivation. The deactivated sulfated zirconia catalyst could be completely regenerated in O$\sb2$ and in air at 450$\sp\circ$C by selectively burning off the coke. Changes in the surface structure of sulfated zirconia were studied using nitrogen adsorption, XRD, TGA and in-situ DRIFTS following deactivation and regeneration of the catalyst. The physical properties of sulfated sol-gel zirconia were unchanged as a result of reaction and catalyst regeneration. Both Bronsted and Lewis acid sites as determined using pyridine adsorption at 100$\sp\circ$C were observed on sulfated zirconia. Only Lewis acid sites were observed on non-sulfated zirconia. The ratio of Bronsted to Lewis acid sites was found to be strongly dependent on catalyst pretreatment temperature prior to reaction. The Bronsted to Lewis acid ratio was observed to decrease from a value of 0.466 to 0.127 following the removal of the catalytically active surface sulfur. The total catalyst acidity was observed to decrease only slightly following removal of this surface sulfur species. An infrared band, centered at 1370 cm$\sp{-1}$, was observed for the catalytically active material. This band appeared following activation in N$\sb2$ at 375$\sp\circ$C and was assigned to S=O. An in-situ DRIFTS accessory and heatable sample cell was designed and constructed. The cell and accessory are simple, ease to make, and inexpensive. The optical system consists of only one spherical mirror and two flat mirrors. The specular reflectance from the optical lense was excluded by manipulating the mirrors such that reflectance by the window did not enter the detector
acase@tulane.edu