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Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The investigation was aimed to improve the understanding of the binding interactions between DevS and DevR that are implicated in the regulation of the dormancy response in Mycobacterium tuberculosis. These binding interactions could provide good drug targets for the treatment of persistent tuberculosis, the mechanistic understanding of their binding interactions is important for the development of a validated inhibitor screen. A detailed in silico analysis of the amino acid residues that play a role in the binding of receptor DevR to both kinase DevS and the target DNA was undertaken. A reasonable approximation of the DevS structure was produced using homologous protein structures. In silico docking of DevS to DevR merely produced a set of probable candidate structures, since more than one conformation with similar docked energies was observed. The decision on which one is the more correct form can only be estimated by crystallization of this complex. Therefore, the functional expression and purification of the Dev TCS components were pursued. Denaturing HIS™-select nickel affinity gel purification in the form of matrix-assisted refolding led to the production of functional Dev TCS proteins. To understand the binding of DevR to DNA consensus sequences, as well as the nature of these interactions, a model was built of the full length DevR dimer binding to DNA consensus sequences. Based on this model, single mutations were made to DevR in vitro and their effects assessed in order to validate the model built. During Electrophoretic Mobility Shift Assay (EMSA) analysis, it was found that K179I and N183L mutants prevented the binding of DevR to the DNA consensus sequences. If DevR and DevS binding are to be used in a drug development program, it is essential to have the protocols to accurately measure their interaction, in addition to developing a fundamental understanding of how their interactions occur. The binding affinity of DevR to both DevS and the truncated soluble fragment of DevS (DevS201) were explored, using the BIAcore instrument, an SPR-based biosensor. For sufficiently strong binding between a histidine kinase and a response regulator, the KD needs to be in the nM range. The KD was calculated to be 255 nM for DevS201 and 184 nM for DevS. Therefore it can be concluded that DevS201 binds DevR strongly enough to be used in future studies, and that the BIAcore could be used to screen small-molecule inhibitors of DevR-DevS interactions. / AFRIKAANSE OPSOMMING: Die Dev twee komponent sisteem (TKS) bestaan uit ‘n histidine kinase naamlik (DevS) en ‘n reaksie reguleerder DevR. DevS en DevR is betrokke by die regulering van die dormante stadium van Mycobacterium tuberculosis. Hierdie meganisme kan ‘n deurbraak dwelm teiken vir die behandeling van sluimerende tuberkulose wees. Die meganisme van hierdie bindings interaksies is van kritieke belang, tesame met die ontwikkeling van 'n erkende inhibeerder toets. ‘n Gedetaileerde in silico analise van die aminosuur volgordes wat 'n rol speel in die binding van die reseptor DevR aan beide DevS sowel as die teiken DNS is voltooi. ‘n Model van die DevS struktuur is saamgstel met behulp van homoloë proteïen strukture. In silico mering van DevS aan DevR het `n stel van die waarskynlike kandidaat strukture verskaf, aangesien meer as een konformasie met soortgelyke merings energieë waargeneem is. Die mees waarskynlike vorm kan alleenlik geïndentifiseer word na kristallisasie van hierdie kompleks. Die funksionele uitdrukking en suiwering van die Dev TKS proteine is gevolglik uitgevoer. Funksionele Dev TKS proteïene is verkry deur denaturerende HIS-select nikkel affiniteit jel suiwering, in die vorm van matriks-geassisteerde hervouing te gebruik. Ten einde die binding te verstaan tussen DevR en DNS konsensus volgordes, sowel as die aard van hierdie interaksies, is 'n model gebou van die volle lengte DevR dimeer binding aan DNS konsensus volgordes. Hierdie model is gevalideer deur punt mutasies in DevR te skep en die gevolge daarvan te beoordeel met elektroforetiese mobiliteits verskuiwing reaksie analises. Dit is bevind dat K179I en N183L mutante, verhoed die binding van DevR aan die DNS konsensus volgordes. Die gebruik van DevR en DevS bindings in ‘n dwelm ontwikkelingsprogram, benodig die fundamentele begrip van hoe die interaksies plaasvind, sowel as akkurate protokolle om die interaksies te meet. Die BIAcore instrument, ’n SPR-gebaseerde biosensor, is ingespan om die bindings affiniteit van DevR aan beide DevS en die fragment van DevS (DevS201) te ondersoek. Om voldoende sterk binding tussen DevS en die DevR te verseker, moet die KD in die nM omgewing wees. Die KD is bepaal as 255 nM en 184 nM vir DevS201 en DevS, onderskeidelik. Die afleiding kan dus gemaak word dat DevS201 sterk genoeg aan DevR bind om in verdere studies gebruik te kan word, en dat die BIAcore gebruik kan word om klein-molekule inhibeerders van DevR-DevS interaksies te toets.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction – Due to spatial separation from the native vascular bed, solid tumours develop regions with limited access to nutrients essential for growth and survival. The promotion of a process known as macroautophagy may facilitate in the maintenance of intracellular amino acid levels, through breakdown of cytoplasmic proteins, so that they remain available for macromolecular biosynthesis and ATP production. Several studies point to the potential ability of some cancers to temporarily increase autophagy and thereby prolong cell survival during metabolic stress. The validity of these claims is assessed when a commonly used breast cancer cell line and an epithelial breast cell line are starved of amino acids in this study. Furthermore, we go on to hypothesize that acute amino acid deprivation during treatment will result in an elevated sensitivity of MDAMB231 cells to doxorubicin toxicity but limit its cytotoxic side-effects in MCF12A cells. Methods and study design- Human breast cancer cells (MDAMB231) and breast epithelial cells (MCF12A) cultured in complete growth medium were compared to those incubated in medium containing no amino acids. Steady state autophagy levels were monitored using classical protein markers of autophagy (LC3-II and beclin-1) and the acidic compartmentalization in cells (Lysotracker™ red dye) in conjunction with autophagy inhibition (bafilomycin A1 and ATG5 siRNA). Cell viability was monitored using several techniques, including caspase 3/7 activity. ATP levels were assessed using a bioluminescent assay, while mass spectrometry based proteomics was used to quantify cellular amino acid levels. Similar techniques were used to monitor autophagy during doxorubicin treatment, while cellular doxorubicin localization was monitored using immunofluorescence microscopy. Finally, a completely novel GFP-LC3 mouse tumour model was designed to assess autophagy and caspase activity within tumours in vivo, during protein limitation and doxorubicin treatment. Results - Amino acid deprivation resulted in a transient increase in autophagy at approximately 6 hours of amino acid starvation in MDAMB231 cells. The amino acid content was preserved within these cells in an autophagy-dependent manner, a phenomenon that correlated with the maintenance of ATP levels. Inhibition of autophagy during these conditions resulted in decreased amino acid and ATP levels and increased signs of cell death. MCF12A cells displayed a greater tolerance to amino acid starvation during 24 hours of amino acid starvation. Evidence indicated that autophagy was important for the maintenance of amino acid and ATP levels in these cells and helped prevent starvation-induced cell death. Furthermore, data showed that concomitant amino acid withdrawal resulted in decreased cellular acidity in MDAMB231 cells, and increased acidity in MCF12A cells, during doxorubicin treatment. These changes correlated with evidence of increased cell death in MDAMB231 cells, but a relative protection in MCF12A cells. A novel model was used to apply these techniques in vivo, and although mice fed on a low protein diet during high dose doxorubicin treatment had increased mean survival and smaller tumour sizes, evidence suggested that autophagy is protecting a population of cells within these tumours. Conclusions - This novel approach to tumour sensitization could have several implications in the context of cancer therapy, and given the delicate relationship that autophagy has with the cancer microenvironment, efforts to determine the mechanisms involved in autophagy and sensitization could lead to new and innovative treatment opportunities for cancer management. / AFRIKAANSE OPSOMMING: Inleiding – As gevolg van hul skeiding van die oorpronklike vaskulêre netwerk, ontwikkel soliede gewasse areas met beperkte toegang tot noodsaaklike voedingstowwe. Die bevordering van 'n proses wat as makro-autofagie bekend staan, kan die handhawing van intrasellulêre aminosuur vlakke fasiliteer. Voorafgenoemde proses word waarskynlik deur die afbreek van sitoplasmiese proteïene teweegebring om sodoende vir makro-molekulêre biosintese en ATP produksie beskikbaar te kan wees. Verskeie studies dui daarop dat sommige kankersoorte die vermoë het om autofagie tydelik te verhoog, en daarby sel oorlewing gedurende metaboliese stress te verleng. Die geldigheid van hierdie eise word evalueer wanneer 'n algemeen beskikbare borskanker sellyn, en 'n borsepiteelsellyn in hierdie studie van aminosure verhonger word. Verder, veronderstel ons dat akute aminosuur ontneming gedurende behandeling 'n verhoogde sensitiwiteit van MDAMB231 selle tot doxorubicin toksisiteit tot gevolg sal hê, maar terselfdetyd die middel se sitotoksiese newe-effekte in MCF12A selle sal beperk. Metodes en studie ontwerp – Menslike borskanker- (MDAMB231) en bors epiteel selle (MCF12A) wat in volledige groeimedium gekweek is, is vergelyk met selle wat in aminosuur vrye medium gekweek is. Basislyn autofagie-vlakke is gemonitor deur die gebruik van klassieke autofagie proteïen merkers (LC3-II en beclin-1) en die asidiese kompartementalisering in selle (Lysotracker™ rooi kleurstof) saam met autofagie inhibisie (bafilomycin A1 and ATG5 siRNA). Sellewensvatbaarheid is deur die gebruik van verskeie tegnieke, insluitend caspase 3/7 aktiwiteit, gemonitor. ATP-vlakke is deur die gebruik van 'n bioluminiserende tegniek gemeet, terwyl massa-spektrometrie-gebaseerde “proteomics” gebruik is om sel aminosuur vlakke te kwantifiseer. Soortgelyke tegnieke is gebruik om autofagie gedurende doxorubicin behandeling waar te neem, terwyl sellulêre doxorubicin lokalisasie deur die gebruik van immunofluoresensie mikroskopie gemonitor is. Ten slotte, is 'n unieke GFP-LC3 muismodel in hierdie studie ontwikkel. Hierdie model is gebruik om autofagie en caspase aktiwiteit in gewasse in vivo te bestudeer tydens proteïen beperking en doxorubicin behandeling. Resultate – Aminosuur ontneming het tot 'n tydelike verhoging in autofagie na ongeveer 6 ure van aminosuur verhongering in MDAMB231 selle gelei. Die aminosuur inhoud van hierdie selle het op 'n autofagie-afhanklike manier behoue gebly. Hierdie verskynsel het met die handhawing van ATP-vlakke gekorreleer. Autofagie inhibisie gedurende hierdie kondisies het 'n verlaging in aminosuur en ATP-vlakke teweeggebring, sowel as vermeerderde tekens van seldood tot gevolg gehad. MCF12A selle het 'n groter toleransie tot aminosuur verhongering tydens die 24 uur aminosuur verhongeringsperiode getoon. Getuienis het aangedui dat autofagie belangrik vir die handhawing van aminosuur en ATP-vlakke in hierdie selle was, en gehelp het om verhongerings-geïnduseerde seldood te voorkom. Verder het data gewys dat aminosuur ontrekking tot verminderde sellulêre asiditeit in MDAMB231 selle, en verhoogde asiditeit in MCF12A selle gedurende doxorubicin behandeling gelei het. Hierdie veranderinge stem ooreen met getuienis van toenemende seldood in MDAMB231 selle, maar 'n relatiewe beskerming in MCF12A selle. 'n Unieke model was gebruik om hierdie tegnieke in vivo toe te pas. Alhoewel verhoogde oorlewing en kleiner gewasse in muise op 'n lae proteïen dieet gedurende hoë dosis doxorubicin behandeling opgemerk is, het bewyse voorgestel dat autofagie 'n populasie selle binne die gewasse beskerm. Gevolgtrekkings – Hierdie unieke benadering tot tumor sensitisering kan verskeie implikasies in die konteks van kanker behandeling hê. Gegewe die delikate verhouding van autofagie met die kanker mikro-omgewing, kan pogings om die meganismes betrokke in autofagie en sensitisering te bepaal, tot nuwe en innoverende behandelings vir kanker lei.

Using three complementary approaches, this work sought to tackle the widespread problem of antibiotic resistance. To circumvent the resistance mechanisms developed by bacteria, it is necessary to establish drug candidates that act on novel therapeutic targets, such as the ion channels used by bacteria to modulate homeostasis. Examples include the potassium efflux channel, Kef, and the mechanosensitive channel of small conductance, MscS, which are not found in humans. How these targets function must be well understood before drug candidates can be developed, as such, their identification and investigation is often accompanied by the evolution of the analytical techniques used to study them. Membrane protein mass spectrometry is one technique showing potential in the study of ion channels. However, spectra can be clouded by the detergents used to solubilise ion channels from their native membranes. Undertaken herein was the synthesis of some fluorescent glycolipid detergents, which it was hypothesised could be encouraged to dissociate from ion channels via laser-induced excitation within the gas phase of a mass spectrometer, thereby improving the clarity with which spectra can be obtained. For Kef, an unconfirmed mechanism of action had previously been proposed. To explore the suggestion that sterically-demanding central residues are important for channel activation, solid phase peptide synthesis was used to isolate three tripeptide analogues of N-ethylsuccinimido glutathione, a known activator with a high affinity for Kef. A competition fluorescence assay suggested these tripeptides bound to Kef with an affinity lower than predicted, allowing the conclusion that a more detailed assessment of the steric bulk required for activation was necessary before a mechanism of action could be confirmed. Lysophosphatidylcholine has been shown to activate MscS, although it is not known how. Affinity chromatography between MscS and lysophosphatidylcholine was proposed as a means by which specific binding interactions could be investigated. For this technique an amino-derivative of lysophosphatidylcholine was necessary and its challenging synthesis is also detailed herein.

Ce travail de thèse est consacré à la synthèse et la caractérisation structurale par spectroscopies RMN, IR, CD et RX de nouveaux oligomères non naturels qui s'inscrivent dans la famille des foldamères. Ils sont construits en particulier à partir d'un β-aminoacide bicyclique chiral original: l'acide (S)- ou (R)-aminobicyclo[2.2.2]octane-2-carboxylique ((S)- et (R) ABOC) que nous avons développé. Ce motif est issu d'une synthèse stéréocontrollée, dont l'étape clef est une réaction de Diels-Alder asymétrique. Après avoir montré que ce motif est un mime de coude peptidique il a été utilisé pour la synthèse de plusieurs séries d'oligomères. Deux grandes familles ont été développées: des oligourées et des oligoamides. A l'intérieur de ces familles, des homo- et des hétéro-oligomères ont été synthétisés et leur préférence conformationnelle a été définie. Les analyses RMN et les études cristallographiques ont permis de montrer qu'en fonction de leur séquence ils sont capables d'adopter plusieurs types d'hélices. Les homo-oligourées d'ABOC et des hétéro-oligourées alternant l'ABOC et des β3-aminoacides ont conduit à des hélices-12/14. Les oligoamides construits par des alternances d'ABOC et de β3-aminoacides dans un rapport 1/1 et des alternances d'ABOC et d'α-aminoacides dans un rapport 1/1 ou 1/2 ont conduit respectivement à des hélices 10/12, 16/18 et 12/14. / This thesis is devoted to the synthesis and FT-IR, CD, NMR and X-ray structural characterization of new unnatural oligomers belonging to the family of foldamers. In particular they are constructed from an original chiral bicyclic β-amino acid: (S)- and (R)-aminobicyclo[2.2.2]octane-2-carboxylic acid ((S) - and (R) ABOC ) that we have developed. This motive was obtained via a stereocontrolled synthesis using an asymmetric Diels-Alder reaction as key step. Firstly, this motive was shown to induce turn in peptide sequence and then it was used for the synthesis of several series of oligomers. Two main families were developed: oligoureas and oligoamides. Within these families, homo-and hetero-oligomers were synthesized and their conformational preferences were defined. NMR analysis and crystallographic studies have shown that depending on their sequence they are able to adopt several types of helices. ABOC homo-oligoureas and hetero-oligoureas containing both ABOC residue and β3-amino acid favor a 12/14-helix. Oligoamides with 1/1 alternation of ABOC and β3-amino acids, and heterogeneous backbones with 1/1 and 1/2 ABOC/α-amino acid residue patterns adopt 10/12-, 16/18- and 12/14-helix, respectively.

Le tripeptide GPE possède une activité neuroprotectrice intéressante in-vitro et in-vivo pour différents types de dommage neuronaux. L'objectif de cette thèse est de mettre au point des méthodes de couplage peptidique pour les acides aminés trifluorométhylés et de réaliser des analogues trifluorométhylés du GPE.Au cours de cette thèse, la synthèse d'acides aminés trifluorométhylés cycliques énantiopurs (proline, pseudoproline) a été réalisée à l'échelle du gramme.Ensuite, une étude méthodologique de l'incorporation de ces acides aminés trifluorométhylés dans des peptides a été effectuée.Des expériences RMN ont été menées pour étudier la conformation cis/trans de la liaison amide de ces peptides trifluorométhylés.Enfin, des analogues du tripeptides GPE ont été synthétisé en remplaçant le motif proline par une proline ou pseudoproline trifluorométhylé. Des tests biologiques sont actuellement en cours pour évaluer leur activité sur divers troubles neuronaux. / GPE is a tripeptide with neuroprotecting activity both in-vitro and in-vivo for various neuronal dammage. The goal of this thesis was to work out peptide coupling methods for trifluoromethylated amino acids and the synthesis of trifluoromethylated analogs of GPE.During this thesis, the synthesis of enantiopure cyclic trifluoromethylated amino acids (proline and pseudoprolines) was performed successfully on grams scale.Then, methodological study of their incorporation in peptide was performed.NMR experiments were made to investigate the cis/trans conformation of the amide bound of trifluoromethylated peptide. At last, analogs of the tripeptide GPE were made with trifluoromethylated proline and pseudoprolines instead of the proline. Biological tests are currently under progress to evaluate their activity on various neuronal disorders.

Recently, several spectrometers for small volume measurements in order of microliter have been introduced. They are primarily intended for protein determination (or determination of proteins and nucleic acids in one measurement) by direct spectrophotometry or other spectral methods. One of such instruments is the NanoVueTM Plus (GE Healthcare). In this work, we first tried to characterize the instrument in general terms (stability) and to optimize measurement condititions (sample volume). Proteins have been determined by direct spectrophotometry using internal programs of the instrument, data were controlled by an independent computation. We studied also influence of differences in composition of various proteins on the results. According to the results of this Thesis, the most accurate values could be obtained using the internal program E 1%, using the E 1% value from an experiment. On the other hand, the program Protein UV is producing often inaccurate values, strongly infleunced by the protein amino acid composition. Keywords: protein determination, spectrophotometer NanoVueTM Plus, influence of amino acid composition

Conformationally constrained bicyclic amino acids are invaluable in the synthesis of natural products and peptidomimetics. Aeruginosins contain novel bicyclic amino acid, 2--‐carboxy--‐6--‐hydroxyl octahydrindole (Choi) as the core structure. Aeruginosins are tetra peptide serine protease inhibitors isolated from marine sponges and cyanobacterial water blooms. Rigid bicyclic amino acid(Choi)is an essential core structure, which strongly influences biological activity of aeruginosin family members. Aeruginosins showed promising inhibitory activity against thrombin, trypsin, and factor VIIa. Thrombin and factor VIIa play a major role in blood clotting cascade; excessive coagulation leads to thrombosis and other cardiovascular diseases. Several research groups have reported a number of synthetic aeruginosin analogs. In this thesis, some of the synthetic methodologies of bicyclic amino acid core of aeruginosins are presented. Importance of bicyclic amino acids in peptidomimetic synthesis and drug designing is presented. Mainly, syntheses of ring oxygenated Choi analogs starting from glucose and mannose are presented.

O carcinoma hepatocelular é um dos tipos de cânceres mais comuns em adultos com sua incidência aumentando mundialmente a cada ano. O tratamento curativo é o transplante de fígado, mas as muitas dificuldades encontradas para o procedimento faz com que a quimioterapia seja amplamente utilizada. Além disso, pode ocorrer quimiorresistência e muitos efeitos adversos, o que impulsiona a busca por novos compostos terapêuticos. L-aminoácido oxidases (LAAO) isoladas de peçonhas de serpentes têm demonstrado bons resultados de citotoxicidade em linhagens celulares tumorais, no entanto estes resultados representam sistemas in vitro em monocultura e estudos recentes demonstram que o microambiente tumoral tem um importante papel na transformação neoplásica, no crescimento e invasão do tumor e na resistência quimioterápica. Assim, avaliou-se uma LAAO purificada da peçonha de Bothrops jararacussu (BjussuLAAO-II) em células de carcinoma hepatocelular (HepG2) em monocultura e em cocultura com células endoteliais de veia umbilical humana (HUVEC) com o objetivo de simular o microambiente tumoral, onde, normalmente, é observado mais de um tipo celular. A atividade citotóxica foi avaliada por meio do ensaio do MTT e do ensaio de sobrevivência clonogênica, a atividade genotóxica por meio do ensaio do cometa, a produção de espécies reativas de oxigênio por meio de fluorescência e os danos ao cromossomo por meio do ensaio do micronúcleo. Em células HepG2, em monocultura, todas as concentrações testadas (0,25 - 5,00 ?g/mL) foram citotóxicas e aumentaram os níveis intracelulares de espécies reativas de oxigênio. Verificou-se dano genotóxico na concentração de 5,00 ?g/mL e não houve dano cromossômico. Quando cultivada em cocultura com células HUVEC, as concentrações de 1,00 e 5,00 ?g/mL de BjussuLAAO-II foram citotóxicas às células HepG2 e aumentaram os níveis de espécies reativas de oxigênio. A concentração de 5,00 ?g/mL induziu danos ao DNA e não houve danos aos cromossomos. Estes efeitos podem ser correlacionados ao aumento de espécies reativas de oxigênio intracelulares e as diferenças entre os resultados de mono e cocultura devido à simulação de parte do microambiente tumoral. Em monocultura de células HUVEC, todas as concentrações testadas foram citotóxicas, houve dano genotóxico nas concentrações de 1,00 e 5,00 ?g/mL e nenhuma concentração testada induziu danos cromossômicos. Como há elevada citotoxicidade, danos ao DNA e indução de efeitos pró-oxidantes em células HepG2, BjussuLAAO-II representa um composto promissor no desenvolvimento de novos fármacos antitumorais / Hepatocellular carcinoma is one of the most common types of cancers in adults whose incidence increases worldwide each year. Liver chirurgic and transplantation is the best choice of treatment, but the many difficulties encountered in the procedure cause chemotherapy to be widely used. In addition, chemo resistance and many adverse effects can occur, which improves the search for new therapeutic molecules. L-amino acid oxidases (LAAO) isolated from snake venoms have demonstrated good cytotoxicity results in tumor cell lines, however these results represent in vitro monoculture systems and recent studies have shown that the tumor microenvironment plays an important role in neoplastic transformation in tumor growth and invasion and chemotherapeutic resistance. A LAAO purified from Bothrops jararacussu venom (BjussuLAA-IIO) venom was evaluated in hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with human umbilical vein endothelial cells (HUVEC) with the aim of simulating the tumor microenvironment, where more than one cell type is normally observed. Cytotoxic activity was assessed by the MTT assay and clonogenic survival assay, genotoxic activity by comet assay, reactive oxygen species production by fluorescence, and chromosome damage by the micronucleus assay. In HepG2 cells, in monoculture, all concentrations tested (0.25 - 5.00 ?g/mL) were cytotoxic and increased intracellular levels of reactive oxygen species. Genotoxic damage at the concentration of 5.00 ?g/mL was observed and there was no chromosomal damage. When cultured with HUVEC cells, concentrations of 1.00 and 5.00 ?g/mL of BjussuLAA-II were cytotoxic to HepG2 cells and increased levels of reactive oxygen species. The concentration of 5.00 ?g/mL induced DNA damage and there was no damage to the chromosomes. These effects can be correlated to the increase of intracellular reactive oxygen species and the differences between the mono and co-culture results due to the simulation of part of the tumor microenvironment. In HUVEC monoculture, all concentrations tested were cytotoxic, genotoxic damage at concentrations of 1.00 and 5.00 ?g/mL, and no concentration tested induced chromosome damage. As there is high cytotoxicity, DNA damage and induction of pro-oxidant effects in HepG2 cells, BjussuLAAO-II represents a promising compound in the development of novel antitumor drugs

Triptofano (TRP), um amino ácido essencial, é metabolizado por duas vias principais, a via das quinureninas e a via serotonérgica. Em ambas as vias há a possibilidade de formação de compostos ativos no sistema imune que se caracterizam pelas ações imunossupressoras e indutoras de tolerância. Na via serotonérgica há a formação de serotonina (5-HT) e em alguns tecidos de melatonina (MEL). Este composto pode ainda ser oxidado por ação de peroxidases aos seus produtos de abertura de anel indólico o AFMK (N1-acetil-n2-formil-5-metoxiquinuramina) e AMK (N1-acetil-5-metoxiquinuramina). Já na via das quinureninas, o TRP é diretamente metabolizado à N-formilquinurenina (NFK) e este é rapidamente deformilado a quinurenina (QUIN). Neste projeto avaliamos qual o efeito do choque endotóxico induzido por injeção endovenosa de LPS (1 mg/kg) sobre a biodisponibilidade de TRP e formação de seu metabólito QUIN. Este estudo foi realizado em condições controle e na vigência de sobrecarga de TRP (administração subcutânea de 0,8 mg/kg). Utilizamos ratos machos Wistar com 30 dias separados em quatro grupos: GI (controle), GII (LPS), GIII (TRP) e GIV (TRP+LPS). TRP (0,8 mg/Kg) foi injetado por via subcutânea nos tempos 0 e 2 horas. Quando injetado, LPS (1 mg/kg) foi administrado por via intravenosa no tempo 2 horas. Após 1 hora da última administração, sangue e cérebro foram coletados. O cérebro foi seccionado em três regiões: cerebelo, córtex e mesencéfalo, os quais foram processados para obtenção de homogenatos. Tanto os homogenatos quanto o soro foram tratados com acetona para extração de TRP e seus metabólitos. A análise destes compostos foi realizada por cromatografia líquida de alta eficiência (HPLC). A administração de TRP elevou significativamente a sua concentração no soro e no SNC. Quando da administração de LPS no grupo que já havia recebido sobrecarga de TRP (GIV) houve uma marcada elevação de TRP e de QUIN séricos e das regiões do SNC, especialmente na região do córtex. Concluímos que na vigência de choque endotóxico há um aumento da biodisponibilidade de TRP, tanto no soro como no SNC e que há um aumento da metabolização deste pela rota das quinureninas, possivelmente via IDO. Estes resultados contribuem para a compreensão da toxicidade de TRP, especialmente relevante no caso em que haja um choque endotóxico concomitante e evidencia o córtex como uma região mais susceptível para os efeitos tóxicos do TRP. / Tryptophan (TRP) is an essential amino acid, metabolized by two main paths; the kynurenine and the serotonergic pathways. In both, there is the possibility of generation of biologic active compounds, especially on the immune system leading to immunosuppression and tolerance. In the serotonergic path there is the formation of serotonine (5-HT) and in some tissues of melatonine (MEL). The latter can be oxidized by the action of peroxidases to its indole ring opening product AFMK (N1-acetil-n2-formil-5-methoxikynuramine) and AMK (N1-acethyl-5-methoxykynuramine). In the kynurenine path, TRP is metabolized to N-formylkynurenine (NFK) that is deformilated to kynurenine (KYN). In this study we evaluated the effect of a endotoxic skock induced by an intravenous injection of LPS (1 mg/kg) on the bioavailability of TRP and formation of KYN. This study was carried out in control conditions and on TRP overload (subcutaneous administration of 0,8 mg/Kg). One month old male Wistar rats were divide in four groups: GI(control), GII(LPS), GIII(TRP) and GIV (TRP+LPS). TRP (0,8 mg/kg) was subcutaneously injected at zero and 2h times. When injected, LPS (1mg/kg) was intravenously administered at 2 h. After one hour from the last administration, blood and brain were collected. Brain is separated in cerebellum, midbrain and cortex and was lysed for the preparation of homogenates. Both, serum and homogenates were extracted in acetone; TRP and KYN were analyzed by HPLC. TRP overload caused a significant increase in its concentration in serum and brain. When LPS was administered in conjunction with TRP overload (GIV) there was a remarkable increase in TRP and KYN in serum and brain, especially in cortex. Our conclusion is that in the bioavailability of TRP, in serum and in brain, and its metabolization to kynurenine is increased by inflammation. IDO is probably involved in this condition. Our results contribute to the knowledge of TRP toxicity, particularly with a concomitant inflammation and demonstrate the cortex as a region of more susceptibility to TRP toxicity.