Secoisolariciresinol (SECO) is the major lignan present in flaxseed, but unlike the structurally related lignan nordihydroguaiaretic acid, it is not associated with toxicity. The major phase I metabolite of SECO is lariciresinol, likely formed as a result of para-quinone methide (p-QM) formation followed by an intramolecular cyclization, thereby minimizing any toxicity associated with the p-QM. Four analogues of SECO were used to investigate substituent effects on lignan metabolism and formation of reactive quinones. HPLC methods were developed for analysis of SECO analogues and their metabolites. The stability of SECO analogues (1 mM) in a 50 mM Na2HPO4 buffer at pH 6.0 and 7.4 were quantified. Enzymatic oxidation experiments using mushroom tyrosinase and microsomes harvested from male Sprague-Dawley rats were performed with and without a GSH trapping system. Mass spectrometry and LC-MS were used to identify metabolites. Life Technologies was contracted to perform IC50 inhibition assays on SECO and the SECO analogues against CYP3A4, CYP3A5, CYP2C9 and CYP2C19 cytochrome P450 isoforms. All SECO analogues were stable at pH 6.0. SECO-2 was stable at pH 7.4 but SECO-1, -3 and -4 were unstable at pH 7.4. Autoxidation of SECO -1, -3 and -4 were 1st order reactions with t1/2 of 9.0 h, 1.7 h and 7.0 h respectively. Mushroom tyrosinase oxidations were performed to generate ortho-quinone standards. SECO-1 -3 and -4 were oxidized by mushroom tyrosinase but SECO-2 was not. Trapping with GSH produces aromatic ring conjugates for SECO-1, -3, -4. Results from microsomal oxidations for SECO-1, -3 and -4 are consistent with these standards. SECO-2 was metabolized by a microsomal system to produce a benzyl GSH adduct. Dealkylation products were also observed. All SECO analogues formed quinones but interestingly, GSH conjugation was competitive with intramolecular cyclization. All cytochrome P450 isoforms were inhibited by every analogue tested to varying degrees, a potential cause of toxicity concerns. Quinones are known to cause toxicity in vivo, including cytotoxicity, immunotoxicity, and carcinogenesis. Our results suggest that since the phenol and catechol lignans form GSH adducts in addition to intramolecular cyclization products, this class of lignans have the potential to cause toxicity.

Target-guided synthesis is an approach to drug discovery that uses the biological target as a template to direct synthesis of its own best inhibitors from small molecule fragments. The process bridges the gap between chemical synthesis of drug candidates and their biological binding assay, merging the two operations into a single process whereby the active site or a binding pocket within the structure of the biological target directly controls the assembly of the best inhibitor in situ. Two different approaches to target-guided synthesis, the thermodynamic approach, making use of reversible reactions, and the kinetic approach, which uses an irreversible reaction, have been employed to discover novel, isoform selective inhibitors of the glutathione transferase (GST) enzyme family – possible drug targets in cancer and parasitic disease treatments. The thermodynamic approach described in this thesis uses the aniline-catalysed reversible acyl hydrazone formation reaction to create a dynamic covalent library of bivalent ligands designed to bind the dimeric structure of GST. In the presence of GST one of the bivalent ligands was selectively amplified at the expense of the other library members. This ligand was shown, via biological assays, to be a specific inhibitor for one isoform of GST, the mu isoform mGSTM1-1. A kinetic approach has also been investigated as a way to identify novel bivalent GST inhibitors utilising the Huisgen 1, 3 dipolar cycloaddition reaction. An azide and alkyne fragment library was designed to bind across the dimeric GST structure. The inhibitor structures are therefore bivalent, containing two anchoring fragments known to bind to the GST active site, linked by a triazolopeptide spacer. The triazole provides the click chemistry disconnection, enabling rapid in situ screening of candidate alkyne and azide fragments for inhibitor discovery. Whilst the in situ reaction with GST yielded inconclusive results, a number of the triazole products were found to have low nanomolar inhibitory activity towards GST.

High-risk human papillomaviruses (HPVs) cause 5% of all human cancers worldwide. The HPV capsid consists of 72 disulfide-linked pentamers of major capsid protein L1 and up to 72 molecules of minor capsid protein L2. The viral genome (vDNA) is 8KB circular dsDNA, condensed with histones and complexed with L2. HPV infection requires the virion particle to get access to basal layer keratinocytes, binding and entry of the cells, uncoating, and transport of the viral genomes to the host cell nucleus. During infection, L2 is important for transport of the viral genome from membrane bound vesicular compartments, through the cytosol and into the host cell nucleus. Previous work has identified a conserved disulfide bond between Cys22 and Cys28, which is necessary for HPV16 infection. We hypothesize that endosomal reduction of this disulfide might be important for L2 conformational changes that allow a hydrophobic transmembrane-like region in L2 to span across endosomal membranes, exposing sorting adaptor binding motifs within L2 to the cytosol. Prior research suggests that cytosolic glutathione (GSH) redox potential is important for reduction of disulfide-linked proteins within the lumen of endosomes. This is achieved by endosomal influx of cytosolic reduced cysteine, where it can reduce disulfide bonds in lumenal proteins. Cytosolic GSH regenerates the pool of reduced cysteine needed to maintain endosomal redox potential. Here we studied the relationship between cytosolic GSH and HPV16 infection. siRNA knockdown of critical enzymes of the GSH biosynthesis pathway or the endosomal cystine efflux pump cystinosin caused partial abrogation of HPV16 infection. Likewise, inhibition of the GSH biosynthesis pathway with L-buthionine sulfoximine (L-BSO) blocked HPV16 infection in multiple cell types, suggesting that cytosolic GSH redox may be important for HPV16 infection. Further studies have revealed that the decrease of HPV16 infection is not because of defects in binding, entry, L2 cleavage or capsid uncoating, but rather is due to inefficient cytosolic translocation of L2/viral genome from the trans-Golgi network (TGN). Contrary to our initial hypothesis, we show that L2 is able to span the endosomal membrane and direct TGN localization in the presence of BSO. Lack of cytosolic GSH causes L2/viral genome to become trapped in the TGN lumen. This suggests that there are redox-sensitive viral or cellular factors necessary for L2/viral genome translocation at the TGN. Future research will focus on the redox state of the Cys22-Cys28 disulfide bond during infection of normal and GSH-depleted cells.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by neurofibrillary tangles, senile plaques and loss of synapses. Many studies support the notion that oxidative stress plays an important role in AD pathogenesis. Previous studies from our laboratory employed redox proteomics to identify oxidatively modified proteins in the AD inferior parietal lobule (IPL). The proteins were consistent with AD pathology and have been central to further investigations of the disease. The present study was focused on the identification of specific targets of protein S-glutathionylation in AD, early AD (EAD), and mild cognative impairment (MCI) using a redox proteomics approach. In AD IPL we identified deoxyhemoglobin, α-crystallin B, glyceraldehyde phosphate dehydrogenase (GAPDH), and α-enolase as significantly S-glutathionylated relative to these brain proteins in control IPL. Both, GAPDH and α-enolase were also shown to have reduced activity in the AD IPL. With further investigation gammaenolase, dimethylarginine dimethylaminohyrdolase (DDAH), Cathepsin D, and 14-3-3 gamma were identified as significantly S-glutathionylated in the EAD IPL. Alpha enolase was also identified as significantly S-glutathionylated in MCI IPL. These results provide a correlation in proteins S-glutathionylated in the progression of AD even in the reversible conditions of amnestic MCI. Amyloid beta-peptide (1-42) [Aβ(1-42)], one of the main component of senile plaque, can induce in vitro and in vivo oxidative damage to neuronal cells through its ability to produce free-radicals. The aim of this study was to investigate the protective effect of the xanthate, D609, on Aβ(1-42)-induced protein oxidation using a redox proteomics approach. D609 was recently found to be a free radical scavenger and antioxidant. In the present study, rat primary neuronal cells were pretreated with 50 μM of D609 followed by incubation with 10 μM Aβ(1-42) for 24 hours. In the cells treated with Aβ(1-42) alone four proteins that were significantly oxidized were identified: Glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase, malate dehydrogenase, and 14-3-3 zeta. Pretreatment of neuronal cultures with D609 prior to Aβ (1-42) protects all the identified oxidized proteins in the present study against Abeta(1- 42)-mediated protein oxidation. Therefore, D609 may ameliorate the Aβ(1-42)-induced oxidative modification.

Cytosolic glutathione transferases (GSTs) are a large family of enzymes that play an important role in detoxification of xenobiotics. They catalyse the conjugation of the glutathione tripeptide (GSH) to a wide range of toxic electrophilic acceptors. The overall 3D folds and architectures of the catalytic sites of many GSTs are conserved. They are composed of a well conserved glutathione binding site (G-site) and a promiscuous hydrophobic binding site (H-site). The 3D structure and ligand specificity has allowed the sub-classification of the multiple isoforms within the soluble GST superfamily. GSTs are involved in the drug detoxification and so are the target of medicinal chemistry programmes but it has proven difficult to generate isoform-specific inhibitors due to their inherent promiscuity. In this project, Venughopal Bhat (University of Edinburgh, laboratory of Dr. Mike Greaney) and I have explored a new platform to probe enzyme specificity. Protein-directed dynamic combinatorial chemistry (DCC) allows the assembly and amplification of a ligand within the confines of a binding site. DCC was used as a tool to explore the promiscuous H-site of four eukaryotic GSTs. I purified recombinant forms of SjGST, hGST P1-1, mGST M1-1 and mGST A4-4 from E. coli and assayed them with the universal, synthetic GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). Venughopal Bhat prepared a ten-member, thermodynamically-controlled, dynamic combinatorial library (DCL) of acyl hydrazones from a 1-chloro-2-nitrobenzene aldehyde and ten acylhydrazides. This DCL was incubated with each of the four GST isozymes (spanning diverse classes) and distinct amplification effects were observed for SjGST and hGST P1-1. I subsequently carried out several biophysical experiments in an attempt to rank each of the ligands. These experiements, coupled with molecular modelling, provided insight into the basis of the observed selectivity. Bacterial GSTs are thought to play a role in primary metabolism and display a different GSH-conjugation mechanism compared to the eukaryotic GSTs. A recombinant form of the beta-class GST from the pathogenic bacterium Burkholderia cenocepacia was isolated, purified and biochemically characterised. The same ten-member acylhydrazone DCL was interfaced with the bacterial GST which was shown to amplify a hydrophobic library member that shared structural features with the known substrate 2-hydroxy-6-oxo-6-phenyl-2,4-dienoate (HOPDA). With the collaboration of Venughopal Bhat, I attempted to explore the putative active site of a GST-like protein with an unknown function using the same DCL. Although no amplification was observed, a new aldehyde template was suggested for future DCC experiments on this protein. GSTs are widely employed in biotechnology as protein fusion tags to enhance target protein solubility coupled with a facile enzyme assay. Manish Gupta and Juan Mareque-Rivas (University of Edinburgh) used the N-terminal, hexahistidine-tagged SjGST to demonstrate that quantum dots (QDs) coated with nitrilotriacetic acid (NTA) bound to Ni2+ ions can be used to reversibly and selectively bind, purify, and fluorescently label a His6-tagged GST in one step with retention of enzymatic activity. For this prupose, I purified and characterized both the untagged and hexahistidinetagged – SjGST prior to their experiments.

La structure de trois oxydoréductases (la glutathion peroxydase (Gpx), la thiorédoxine (Trx) et la glutarédoxine (Grx)) de Populus trichocarpa × deltoides (le peuplier) a été caractérisée par diffraction des rayons X. Les Gpxs forment un groupe d’enzymes qui régulent la concentration des espèces réactives de l'oxygène (ROS) dans les cellules, et qui les protègent des effets d’un stress oxydant. Contrairement à leurs homologues d’origine animale, les Gpxs végétales ne dépendent pas du glutathion (GSH) mais des Trx pour leur fonctionnement. Dans cette étude, j'ai résolu les structures des formes réduite et oxydée de la Gpx5 de peuplier et montré que des changements conformationnels drastiques sont nécessaires pour passer d’une forme à l’autre. Les Trxs régulent diverses protéines cibles par la réduction de leur pont disulfure. Mon objectif était de comprendre le mécanisme catalytique d’une nouvelle isoforme, la PtTrxh4, dont la capacité à accepter des électrons de la Grx a été récemment démontrée. Cette PtTrxh4 contient trois cystéines, la première localisée dans une extension en position N-terminale (Cys4) et deux situées dans le site actif classique (WC1GPC2) de la Trx. Les résolutions des structures de l’enzyme sauvage et du mutant C4S m’ont permis de proposer un mécanisme catalytique en quatre étapes en accord avec les études enzymatiques. Les Grxs sont des protéines qui utilisent des électrons du GSH en particulier pour catalyser des réactions d'échange de thiol-disulfure. Ici, je présente la structure de la PtGrxS12 (en complexe avec le GSH), la première structure de la Grx végétale de sous-classe 1 ayant un site actif de motif atypique 28WCSYS32. / Three oxidoreductases (glutathione peroxidase, GPX; thioredoxin, Trx and glutaredoxin, Grx) from Populus trichocarpa × deltoides (poplar tree) were characterized using X-ray crystallography approach. GPXs are a group of enzymes that regulate the levels of oxygen species in cells, and protect them against oxidative damage. In this study, I have determined the crystal structures of the reduced and oxidized form of poplar GPX5 (PtGPX5). Comparison of both redox structures indicates that a drastic conformational change is necessary to bring the two distant cysteine residues together to form an intramolecular disulfide bond. Trxs regulate various protein partners through the thiol-disulfide(s) reduction. The aim of this study is thus to precisely describe the catalytic mechanism of a new isoform of Trx, PtTrxh4, since it has been demonstrated recently to be reduced by Grx. PtTrxh4 contains three cysteines; one localized in an N-terminal extension (Cys4) and two in the usual Trx active site (WC1GPC2). Two crystal structures of PtTrxh4 solved in this study, wild-type and C61S mutant, allow us to propose a four-step disulfide cascade catalytic mechanism in accordance with enzymatic studies. Grxs are highly conserved redox-proteins that utilize electrons from GSH particularly to catalyze thiol-disulfide exchange reactions. Here, I present the structure of glutathionylated PtGrxS12, the first structure of plant Grx of subclass 1 with an atypical 28WCSYS32 active site. Protein structures solved here shed lights to our understanding of the redox mechanism in plant and to the enzyme-substrate interactions.

Student Number : 0002482E - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Proteins in the glutathione transferase family share a common fold. The close packing of secondary structures in the thioredoxin fold in domain 1 forms a compact hydrophobic core. This fold has a bababba topology and most proteins/domains with this fold have a topologically conserved isoleucine residue at the N-terminus of a-helix 3. Class Alpha glutathione transferases are one of 12 classes within the glutathione transferase family. To investigate the role of the conserved isoleucine residue in the structure, function and stability of glutathione transferases, homodimeric human glutathione transferase A1-1 (hGST A1-1) was used as a representative of the GST family. Ile71 was replaced with valine and the properties of I71V hGST A1-1 were compared with those of wildtype hGST A1-1. The spectral properties monitored using far-UV CD and tryptophan fluorescence indicated little change in secondary or tertiary structure confirming the absence of any gross structural changes in hGST A1-1 due to the incorporation of the mutation. Both wildtype and mutant dimeric proteins were determined to have a monomeric molecular mass of 26 kDa. The specific activity of I71V hGST A1-1 (130 mmol/min/mg) was three times that of wildtype hGST A1-1 (48 mmol/min/mg). I71V hGST A1-1 showed increased kinetic parameters compared to wildtype with a 10-fold increase in kcat/Km for CDNB. The increase in Km of I71V hGST A1-1 suggests the mutation had a negative effect on substrate binding. The DDG for transition state stabilisation was –5.82 kJ/mol which suggest the I71V mutation helps stabilise the transition state of the SNAR reaction involving the conjugation of reduced glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). A 2-fold increase in the IC50 value for I71V hGST A1-1 (11.3 mM) compared to wildtype (5.4 mM) suggests that the most noticeable change due to the mutation occurs at the H-site of the active site. Conformational stability studies were performed to determine the contribution of Ile71 to protein stability. The non-superimposability of I71V hGST A1-1 unfolding curves and the decreased m-value suggest the formation of an intermediate state. The conformational stability of I71V hGST A1-1 (16.5 kcal/mol) was reduced when compared to that of the wildtype (23 kcal/mol). ITC was used to dissect the binding energetics of Shexylglutathione to wildtype and I71V hGSTA1-1. The ligand binds 5-fold more tightly to wildtype hGST A1-1 (0.07 mM) than I71V hGST A1-1 (0.37 mM). The I71V mutant displays a larger negative DCp than wildtype hGST A1-1 (DDCp = -0.41 kJ/mol/K). This indicates that a larger solvent-exposed hydrophobic surface area is buried for I71V hGST A1-1 than for wildtype hGST A1-1 upon the binding of S-hexylglutathione. Overall the results suggest that Ile71 conservation is for the stability of the protein as well as playing a pivotal indirect role in catalysis and substrate binding.

Introdução: O exercício resistido em nível atlético pode promover estresse oxidativo crônico, fato que implica em uma resposta imuno-inflamatória exacerbada com consequente redução de desempenho e efeitos à saúde. Ao mesmo tempo, exercícios de caráter intenso elevam o consumo de glutamina por células e tecidos, reduzindo assim a disponibilidade deste aminoácido ao organismo, Todavia, estudos avaliando o metabolismo da glutamina em exercício do tipo resistido ainda são escassos. A síntese de antioxidantes, tais como a glutationa (GSH) e proteínas citoprotetoras como proteínas de choque térmico (HSPs) podem ser influenciadas pela disponibilidade de glutamina. Objetivo: Avaliar o efeito da suplementação oral crônica com L-glutamina e L-alanina, ambas na forma livre ou como Dipeptídeo sobre o estresse oxidativo e citoproteção mediado pela HSP 27 em ratos submetidos a exercício resistido. Métodos: Cinquenta ratos Wistar machos adultos (n = 10 por grupo) foram distribuídos em 5 grupos experimentais: Sedentário (SED), Treinado (CTRL) e suplementados com DIP, solução com L-glutamina e L-alanina livres (GLN+ALA) e somente L-Alanina (ALA), e foram submetidos ao protocolo de subida em escada durante 6 semanas, suplementados na água de beber em uma solução à 4%, nos últimos 21 dias do experimento. Foram analisados: teste de carga máxima, lactato sanguíneo, glutamina e glutamato (plasma, fígado e músculo -Tibial e EDL), creatina kinase e mioglobina (plasma) transaminases (plasma), glutationa oxidada (GSSG) e reduzida (GSH) (papa de hemácias, fígado e músculo - Tibial e EDL), TBARS (fígado e músculo- Tibial e EDL) e, expressão de HSP-27 e Glutamina Sintetase (músculo Tibial). Resultados: Os resultados demonstraram que o protocolo de exercício resistido reduziu a concentração de glutamina no músculo (p<0,05), aumentou a razão [GSSG/GSH] no fígado, papa de hemácia e músculo (p<0,05), e consequentemente houve aumento de TBARS nos tecidos. Já as suplementações com L-glutamina e L-alanina livres e como dipeptídeo aumentaram as concentrações de glutamina no plasma e tecidos (p<005), melhoraram a razão de [GSSG/GSH] no fígado, papa de hemácias e músculo (p<0,05). Também foi encontrado aumento da expressão de HSP 27 no Tibial, redução de TBARS nos tecidos, e creatina kinase no plasma (p<0,05). Conclusão: As suplementações com L- glutamina e L- alanina livres ou como dipeptídeo aumentam a síntese de GSH e a expressão de HSP 27, atenuando assim o estresse oxidativo causado pelo exercício resistido. / Introduction: Athletic Resistance exercise way promotes chronic oxidative stress, which implies in exacerbated immune inflammatory response with consequent reduction in performance and health effects. At the same time, intense exercise improves consumption of glutamine for cells and tissues, thereby reducing the availability of this amino acid to the body. However, studies evaluating the glutamine metabolism in resistance exercise are still scarce. The synthesis of antioxidants such as glutathione (GSH) and cytoprotective proteins such as heat shock proteins (HSPs) can be influenced by the availability of glutamine. Objective: To evaluate the effect of chronic oral supplementation with L-glutamine and L-alanine, both in its free form or as dipeptide on oxidative stress and the cytoprotection mediated by HSP 27 in rats subjected to resistance exercise. Methods: Fifty (n = 10 per group) Wistar adult rats were divided into 5 groups: Sedentary (SED), Trained (CTRL) and supplemented with DIP, solution with L-glutamine and free L-alanine (GLN + ALA) and L-alanine (ALA). The trained groups were underwent to climb stairs protocol for six weeks. Supplementations were offered in 4% solution in drinking water in the last 21 days of the experiment. Were analyzed: maximum load test, blood lactate, glutamine and glutamate (in plasma, liver and muscle Tibialis and EDL), creatine kinase and myoglobin (plasma), transaminase (plasma), oxidized (GSSG) and reduced (GSH) glutathione (erythrocytes, liver and muscle- Tibialis and EDL), TBARS (liver and muscle Tibialis- and EDL), HSP-27 and Glutamine Synthetase expression (Tibialis muscle). Results: The results showed that resistance exercise protocol reduced glutamine concentration in muscle (p <0.05) increased the ratio [GSSG / GSH] in the liver, erythrocytes and muscle (p <0.05), and TBARS increase the tissue. The Supplementation with L-glutamine and L-alanine and free dipeptide and increased glutamine concentrations in the plasma and tissues (p <0.05), improved the ratio of [GSSG / GSH] in liver, erythrocytes and muscle (p <0.05). HSP 27 expression was also increased in Tibialis Muscle. There was reduction of TBARS in tissues and creatine kinase in plasma (p <0.05). Conclusion: Supplementations with L-glutamine and L-alanine in its free form or as dipeptide increase the synthesis of GSH and Expression HSP 27, thus reducing oxidative stress caused by resistance exercise.

by Kwan, Leo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 108-116). / Abstract --- p.i / Achnowledgment --- p.ii / List of abbreviations --- p.iii / List of table and figures --- p.v / Table of contents --- p.vi / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- THE DISCOVERY OF TUMOUR NECROSIS FACTOR (TNF) --- p.1 / Chapter 1.2 --- THE MOLECULE AND ITS RECEPTORS --- p.1 / Chapter 1.3 --- THE BIOLOGICAL ACTIVITIES OF TNF --- p.3 / Chapter 1.4 --- STUDIES ON THE CYTOTOXIC MECHANISM OF TNF --- p.4 / Chapter 1.5 --- A TENTATIVE MECHANISM OF TNF CYTOTOXICITY --- p.11 / Chapter 1.6 --- THE GLUTATHIONE SYSTEM : A CELLULAR PROTECTIVE MECHANISM AGAINST OXIDATIVE STRESS …… --- p.12 / Chapter 1.7 --- OBJECTIVE AND STRATEGY OF THIS STUDY --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND APPARATI / Chapter 2.1 --- CELL LINES --- p.19 / Chapter 2.2 --- "ISOLATION, MAINTENANCE AND SUBCULTURE OF CELL LINES" --- p.19 / Chapter 1. --- Plain RPMI-1640 medium / Chapter 2. --- Penicillin-streptomycin solution / Chapter 3. --- Foetal bovine serum / Chapter 4. --- Complete RPMI-1640 medium / Chapter 5. --- Trypsin-ethylenediaminetetraacetate solution / Chapter 6. --- Phosphate buffered saline / Chapter 7. --- Cycloheximide / Chapter 8. --- Actinomycin D / Chapter 9. --- Trypan blue stain / Chapter 10. --- Neutral red stain / Chapter 11. --- Recombinant human tumour necrosis factor / Chapter 12. --- Cell culture plates and flasks / Chapter 2.3 --- GROWTH CHARACTERISTIC --- p.22 / Chapter 1. --- Tritiated Thymidine / Chapter 2. --- Tritiated Leucine / Chapter 3. --- Trichloroacetic acid / Chapter 4. --- Scintillation cocktail / Chapter 2.4 --- "RESPONSE TOWARDS ANTICANCER DRUGS, CYTOTOXIC AGENTS, AND ENZYME MODULATORS" --- p.23 / Chapter 1. --- N-acetyl-DL-homocysteinethiolactone / Chapter 2. --- Diethyldithiocarbamic acid / Chapter 3. --- Doxorubicin / Chapter 4. --- Acivicin / Chapter 5. --- Ethacrynic acid / Chapter 6. --- "L'Buthionine-[S,R]-sulfoximine" / Chapter 7. --- Hydrogen peroxide / Chapter 8. --- Methotrexate / Chapter 9. --- Menadione / Chapter 2.5 --- CULTURE OF BACTERIAL CELLS --- p.27 / Chapter 1. --- Ampicillin stock solution / Chapter 2. --- Chloramphenicol stock solution / Chapter 3. --- Tetracycline stock solution / Chapter 4. --- Luria-Bertani medium / Chapter 5. --- LB with ampicillin / Chapter 6. --- SOB medium / Chapter 7. --- SOB medium with ampicillin / Chapter 8. --- SOC medium / Chapter 9. --- SB medium / Chapter 10. --- SB medium with ampicillin / Chapter 11. --- Agar plates / Chapter 2.6 --- PREPARATION OF DNA PROBES FROM BACTERIAL CLONES --- p.29 / Chapter 1. --- FlexiPrep Kit / Chapter 2. --- Restriction endonucleases / Chapter 3. --- GeneClean® II Kit / Chapter 4. --- cDNA clones for making DNA probes / Chapter 5. --- TrisHCl EDTA buffer / Chapter 2.7. --- ELECTROPHORESIS OF DNA --- p.31 / Chapter 1. --- EDTA stock solution / Chapter 2. --- Tris acetate EDTA electrophoresis buffer / Chapter 3. --- Tris borate EDTA electrophoresis buffer / Chapter 4. --- Ethidium bromide / Chapter 5. --- DNA molecular size markers / Chapter 6. --- TAE/TBE agarose gel slab / Chapter 2.8 --- CONSTRUCTION OF MURINE TNFR1 PARTIAL cDNA CLONE --- p.33 / Chapter 1. --- Frist strand cDNA synthesis Kit / Chapter 2. --- Murine TNFR1 forward and reverse primers / Chapter 3. --- Polymerase chain reaction reagents / Chapter 4. --- Cloning vector / Chapter 5. --- Modifing enzymes / Chapter 6. --- T7 SequencingTM Kit / Chapter 7. --- Acrylamide/bis gel stock solution / Chapter 8. --- Urea / Chapter 9. --- TEMED and ammonium persulphate / Chapter 10. --- β-Galactosidase colour test reagents / Chapter 11. --- TFB solution / Chapter 12. --- DnD solution / Chapter 2.9. --- RADIOLABELLING OF DNA PROBES --- p.35 / Chapter 1. --- Oligolabelling kit / Chapter 2. --- Redivue [α-32P] dCTP / Chapter 3. --- PUSH column / Chapter 2.10 --- EXTRACTION OF TOTAL RNA FROM CELL LINES --- p.36 / Chapter 1. --- N-Lauroylsarcosine / Chapter 2. --- 2M sodium acetate (pH48) / Chapter 3. --- Phenol / Chapter 4. --- Isopropanol / Chapter 5. --- Ethanol / Chapter 6. --- Extraction buffer / Chapter 7. --- Chloroform / Chapter 8. --- Isoamyl alcohol / Chapter 2.11 --- HYBRIDIZATION AND NORTHERN ANALYSIS --- p.37 / Chapter 1. --- 20XSSC / Chapter 2. --- 5X formaldehyde running buffer / Chapter 3. --- RNA sample buffer / Chapter 4. --- 10X RNA loading buffer / Chapter 5. --- Formaldehyde slab gel / Chapter 6. --- Hybond®-N membrane / Chapter 7. --- Immobilon®-N membrane / Chapter 8. --- Salmon sperm DNA / Chapter 9. --- Sodium dodecyl sulphate / Chapter 10. --- Dextran sulphate / Chapter 11. --- Kodak Biomax MR and X-OMAT films and developing kits / Chapter 2.12 --- APPARATI USED --- p.39 / Chapter CHAPTER 3 --- METHODS / Chapter 3.1 --- ISOLATION AND MAINTENANCE OF TNF RESISTANT L929 CELLS --- p.40 / Chapter 3.1.1 --- Culture of L929 cells / Chapter 3.1.2 --- Trypan blue exclusion test / Chapter 3.1.3 --- Isolation of TNF-resistant variants of L929 / Chapter 3.1.4 --- Verification of the TNF-resistant trait of rL929 / Chapter 3.1.5 --- Neutral red uptake assay / Chapter 3.2 --- COMPARING L929 AND rL929 CELLS IN TERMS OF GROWTH CHARACTERISTICS --- p.43 / Chapter 3.2.1 --- Doubling time / Chapter 3.2.2 --- Rate of protein synthesis / Chapter 3.2.3 --- Rate of DNA synthesis / Chapter 3.3 --- COMPARING L929 AND rL929 CELLS IN TERMS OF RESPONSE TOWARDS DIFFERENT ENZYME INHIBITORS AND CYTOTOXIC AGENTS --- p.44 / Chapter 3.3.1 --- TNF cytotoxicity on L929 and rL929 cells --- p.44 / Chapter 3.3.2 --- Effects of inhibitors of gene transcription and protein synthesis on TNF cytotoxicity on L929 and rL929 cells --- p.44 / Chapter 3.3.3 --- Cytotoxic effect of hydrogen peroxide and menadione on L929 and rL929 cells --- p.44 / Chapter 3.3.4 --- TNF cytotoxicity on L929 and rL929 cells: effect of N-acetyl homocysteine thiolatone --- p.45 / Chapter 3.3.4.1 --- The tolerant limit of AHCT / Chapter 3.3.4.2 --- Effect of AHCT on TNF cytotoxicity / Chapter 3.3.5 --- TNF cytotoxicity on L929 and rL929 cells: effect of diethyldithiocarbamate --- p.46 / Chapter 3.3.5.1 --- The tolerant limit of DEDTC / Chapter 3.3.5.2 --- Effect of DEDTC on TNF cytotoxicity / Chapter 3.3.6 --- TNF cytotoxicity on L929 and rL929 cells: effect of buthionice sulfoximine --- p.47 / Chapter 3.3.6.1 --- The tolerant limit of BSO / Chapter 3.3.6.2 --- Effect of BSO on TNF cytotoxicity / Chapter 3.3.7 --- TNF cytotoxicity on L929 and rL929 cells: effect of Acivicin --- p.47 / Chapter 3.3.7.1 --- The tolerant limit of acivicin / Chapter 3.3.7.2 --- Effect of acivicin on TNF cytotoxicity / Chapter 3.3.8 --- TNF cytotoxicity on L929 and rL929 cells: effect of ethacrynic acid --- p.48 / Chapter 3.3.8.1 --- The tolerant limit of ethacrynic acid / Chapter 3.3.8.2 --- Effect of ethacrynic acid on TNF cytotoxicity / Chapter 3.3.9 --- Cytotoxic effect of doxorubicin on L929 and rL929 cells --- p.49 / Chapter 3.3.10 --- TNF cytotoxicity of L929 cells: effect of N-acetyl cysteine --- p.49 / Chapter 3.3.11 --- Cytotoxic effect of methotrexate on L929 and rL929 cells --- p.50 / Chapter 3.3.12 --- Cytotoxic effect of hyperthermia on L929 and rL929 cells --- p.50 / Chapter 3.4 --- NORTHERN ANALYSIS AND HYBRIDIZATION --- p.51 / Chapter 3.4.1. --- Preparing RNA blots --- p.51 / Chapter 3.4.1.1 --- Extraction of total RNA from cells / Chapter 3.4.1.2 --- Making equal loading of RNA samples in formaldehyde gel electrophoresis / Chapter 3.4.1.3 --- Northern blotting of RNA / Chapter 3.4.2. --- Preparation of cDNA probes --- p.53 / Chapter 3.4.2.1 --- Preparing plasmids from A TCC clones / Chapter 3.4.2.2 --- Preparing TNFR1 probe from first-strand cDNA of L929 cells / Chapter 1. --- Construction of recombinant clone from the PCR product of TNFRl fragment / Chapter 2. --- Transforming the recombinant vector into JM109 host / Chapter 3. --- Sequencing of PCR product for identity confirmation / Chapter 3.4.2.3 --- Preparing DNA inserts from plasmids / Chapter 3.4.3 --- Radiolabelling of cDNA probes --- p.56 / Chapter 3.4.4 --- Hybridization of radioactive probes to RNA blots --- p.57 / Chapter CHAPTER 4 --- RESULTS AND DISCUSSIONS / Chapter 4.1 --- ISOLATION OF TNF-RESISTANT VARIANTS OF L929 CELLS --- p.58 / Chapter 4.1.1 --- Single cell subcloning of TNF-resistant L929 variants / Chapter 4.1.2 --- Growth rates of L929 and rL929 cells / Chapter 4.1.3 --- Rate of protein synthesis in L929 and rL929 cells / Chapter 4.1.4 --- Rate of DNA synthesis in L929and rL929 cells / Chapter 4.2 --- EFFECT OF INHIBITORS OF GENE TRANSCRIPTION AND PROTEIN SYNTHESIS ON TNF CYTOTOXICITY ON L929 AND rL929 CELLS --- p.67 / Chapter 4.3 --- RESPONSE OF L929 AND rL929 CELLS TOWARDS VARIOUS CYTOTOXIC AGENTS --- p.70 / Chapter 4.3.1 --- "Response towards methotrexate, an anti-metabolite used in cancer treatment" / Chapter 4.3.2 --- "Response towards doxorubicin, an chemotherapeutic agent used in cancer treatment" / Chapter 4.3.3 --- "Response towards menadione, a cytotoxic agent known to generate free radicals inside cells" / Chapter 4.3.4 --- Response towards hydrogen peroxide: a highly oxidative agent / Chapter 4.3.5 --- "Response towards hyperthermia, a treatment known to exert oxidative stress on cells" / Chapter 4.4 --- EFFECTS OF MODULATORS OF CYTOSOLIC SUPEROXIDE DISMUTASE ON TNF CYTOTOXICITY ON L929 and rL929 CELLS --- p.77 / Chapter 4.5 --- EFFECT OF MODULATORS OF GLUTATHIONE METABOLISM ON TNF CYTOTOXICITY ON L929 AND rL929 CELLS --- p.82 / Chapter 4.5.1 --- "Effects of L-buthionine [S,R] sulfoximine, an inhibitor of glutathione synthesis" --- p.82 / Chapter 4.5.2 --- "Effect of N-acetyl cysteine, a cysteine derivative" --- p.84 / Chapter 4.5.3 --- "Effects of acivicin , an inhibitor of GSH reuptake and recycle" --- p.85 / Chapter 4.5.4 --- "Effect of ethacrynic acid, an inhibitor of glutathione S- transferase" --- p.87 / Chapter 4.6 --- GENE EXPRESSION IN L929 AND rL929 CELLS IN THE COURSE OF TNF CHALLENGE --- p.89 / Chapter 4.6.1 --- Isolation of total RNA from L929 and rL929 cells --- p.89 / Chapter 4.6.2 --- Preparation of DNA probes for hybridization --- p.89 / Chapter 4.6.3 --- Hybridization of specific probes on RNA blots --- p.90 / Chapter 4.6.3.1 --- Expression of heat shock protein --- p.70 / Chapter 4.6.3.2 --- Expression of the p55 TNF receptor / Chapter 4.6.3.3 --- Expression of glutathione reductase / Chapter 4.6.3.4 --- Expression of glutathione S-transferase pi / Chapter 4.7 --- DISCUSSIONS OF THE EXPERIMENTAL RESULTS --- p.97 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.104 / APPENDIX / Generation of the TNF receptor 1 cDNA probe --- p.106 / REFERENCES --- p.108

As glutationas transferases compõem uma superfamíla de proteínas que atuam na fase II do sistema de desintoxicação das células. Participam principalmente através do processo de conjugação da glutationa com moléculas hidrofóbicas e eletrofílicas, como por exemplo os herbicidas. No entanto, outras funções foram descritas como a tolerância ao estresse oxidativo, inseticidas, antibióticos microbianos, transporte de produtos secundários tóxicos, sinalização da célula durante as respostas ao estresse e fenômenos de resistência envolvendo agentes de quimioterapia contra o câncer. Nesta tese procurou-se estabelecer uma relação entre a seqüência, estrutura, função e afinidade das GSTs. A estrutura, de modo geral, determina a função da enzima, mas por si só, não dita sua especificidade. Esta última informação é fundamental para o desenvolvimento de novos agroquímicos ou para o desenho racional de novas proteínas. A relação entre a seqüência, estrutura, função e afinidade mostra que o paradigma estrutura-função deveria ser ampliado para incluir a seqüência de aminoácidos e a afinidade da enzima. Apesar da grande diversidade de substratos e seqüências encontradas nas GSTs há pelo menos um caso de convergência funcional em duas classes distintas desta superfamília. Uma encontrada apenas no reino Animalia (classe Pi) e outra exclusiva do reino Plantae (classe Phi). Ferramentas da bioinformática estrutural, como docking molecular e minimização de energia foram utilizadas para analisar as interações entre a enzima e o substrato. Estas ajudam a explicar como duas proteínas com aproximadamente 22% de identidade de seqüência apresentam afinidades semelhantes. Finalmente, foram propostos mutantes da GST de Saccharum officinarum utilizando a informação estrutural da enzima, visando uma alteração na afinidade da mesma. / Glutathione Transferases comprehend a superfamily of proteins that plays the phase II of the detoxification system of the cells. Their major catalysis is the conjugation of glutathione with hydrophobic and eletrophilic molecules, for example herbicides. However, other functions were described like oxidative stress, insecticides, microbial antibiotics, transport of secondary products, cells signalization during response to stress and resistance of chemotherapy drugs against cancer. This work aimed to establish a relation between sequence, structure, function and affinity of GSTs. The structure, in general, determines the function, but alone, can not determine the enzyme specificity. This last information is essential to the development of new agrochemicals or for the rational design of proteins. The relation between sequence, structure, function and affinity shows that the paradigm of structure-function should be enlarged in order to include the information of amino acid sequences and the enzyme affinities. Despite the wide variety of substrates and sequences found in the GSTs, there is at least one case of functional convergence between two distinct classes in this superfamily. One is found in the Animalia kingdom (class Pi) and the other is exclusively found in Plantae (class Phi). The structural bioinformatic tools, such as molecular docking and energy minimization were used to analyze the interactions between the enzyme and the substrate. These help to understand how two enzymes with approximately 22% of sequence identity can show the same affinities. Finally, GST mutants of Saccharum officinarum were proposed, using the enzyme structural information in order to modify the enzyme affinities.