Over the past two decades a body of evidence concerning residual biological contamination on cleaned surgical instruments has accumulated. This is substantiated by the number of yearly surgery cancellations due to visible residue on instruments in surgical packs and incidences of iatrogenic Creutzfeldt-Jakob disease (iCJD). It is therefore imperative to develop a method of protein detection for use in clinical sterile services departments (SSDs) for monitoring of decontamination quality. This Thesis describes the development and use of an epifluorescence surface scanner (EFScan) technology in the assessment of proteinaceous residue on surgical instruments, by detecting protein pre-labelled with fluorescein isothiocyanate (FITC), and exploratory studies on the feasibility of label-free detection, using intrinsic protein fluorescence. Measurements using FITC labelling showed that residual protein on the order of micrograms can be found on new, single-use instruments (i.e. prior to use). This is comparable to the amount of residual protein found on retired, reusable instruments. To confirm the suitability of fluorescence techniques in the detection and quantification of proteinaceous residue, a blind, pilot study was carried out in conjunction with groups from Queen Mary University and the University of Southampton. Each University used a different labelling and detection method, and results showed good agreement between these methods. This showed that fluorescence is a suitable technique for the detection and quantification of proteinaceous contamination on surgical instruments. The next step in the project focussed on detection of contamination via intrinsic protein fluorescence from tryptophan residues, with a view to elimination of the labelling step. Intrinsic fluorescence of proteins in solution is widely characterised; however, fluorescence characteristics of solid or surface-bound protein have been little studied. Therefore, the characterisation of solid protein fluorescence and the emission characteristics of protein adsorbed onto stainless steel was undertaken. Analysis of the commonly used protein standard bovine serum albumin (BSA) showed that the two tryptophan residues it contains are highly susceptible to photo-oxidation in the solid state, resulting in conversion to the fluorescent photoproducts n-formylkynurenine (NFK) and kynurenine. Therefore, BSA is not suitable for use as a standard in the development of intrinsic fluorescence detection of surface-bound protein. The 72-tryptophan protein fibrinogen, as well as a series of other multi-tryptophan proteins, were assessed and it was found that photo-oxidation of the tryptophan residues did not occur on the irradiation timescale of 1 hour utilized. Therefore, it was concluded that lysozyme or gamma-globulins, a prominent group of serum proteins, would be more suitable candidates as a standard in subsequent research into the intrinsic detection and quantification of proteinaceous contamination. A third study explored the potential use of fluorescence in the early diagnosis of cataract. This involved the fluorescence characterisation of healthy porcine lenses and the use of UV irradiation of the lens to attempt to create cataract in vitro. There was found to be a large variation in fluorescence characteristics from lens to lens, suggesting that fluorophore concentrations can vary significantly. This implies that identification of a suitable standard for the early detection of cataract may be problematic. Attempts to create cataract resulted in the photo-oxidation pathway which had been observed in BSA, and although NFK and kynurenine play a role in cataractogenesis, the accumulation of these photoproducts is not analogous to a natural cataract. It was found that these products could be destroyed by irradiation of the lens at appropriate photo-bleaching wavelengths. However, this also destroyed intrinsic, protective fluorophores within the lens, suggesting that a light-based method of cataract treatment may not be achievable.

The kynurenine pathway produces NAD+ from L-tryptophan. Metabolites known as the kynurenines are produced within the pathway. The effects of the kynurenines have been associated with a number of diseases including cancer, Alzheimer’s disease, Huntington’s disease, and acute pancreatitis. Kynurenine monooxygenase (KMO) is an enzyme that catalyses the conversion of L-kynurenine to 3-hydroxy-L-kynurenine, the downstream product of which is the neurotoxic quinolinic acid. L-kynurenine is positioned at a branching point within the pathway. Metabolism via KMO leads to quinolinic acid production whereas conversion via kynurenine aminotransferase (KAT) produces the neuroprotective kynurenic acid. Inhibition of KMO leads to an increase in kynurenic acid concentration. This has also been shown to ameliorate the symptoms of neurological diseases in a number of animal models as well as to protect against multiple organ dysfunction caused by acute pancreatitis in rodent models. These findings present KMO as a promising drug target. Due to the hydrophobic nature of human KMO (hKMO) it has been necessary to utilise other forms of KMO as models. Past studies have produced crystal structures of a truncated Saccharomyces cerevisiae KMO and of Pseudomonas fluorescens KMO (PfKMO). Previous work in this research group has resulted in the structure of variants of PfKMO bound to either inhibitor molecules or substrate. These structures identified residues involved in substrate binding and the presence of a highly mobile section of the C-terminus, giving rise to open and closed conformations. It was surmised the movement of the C-terminus was dependent upon the presence of substrate and an interactive network between the C-terminus and the rest of the protein. Using improved crystallising conditions high-resolution structures of PfKMO have been produced that allow for further study of residues involved in substrate binding and the interactive network within the C-terminus. The mutants R84K and Y404F showed severely decreased enzyme activity. Crystal structures of these proteins showed disrupted interactions between substrate and active site. These findings underline the importance of residues R84 and Y404 in substrate binding. An H320F mutation gives an analogous active site to hKMO. Crystallographic and kinetic study of this mutant proved very similar to PfKMO, supporting the use of PfKMO as a model for hKMO. Throughout the work each structure had a P21221 space group with two molecules in the asymmetric unit. The presence of an open and closed molecule within each structure, including substrate-free molecules refuted the connection between C-terminus and substrate. R386K and E372T mutations were separately introduced in order to interrupt the interactive network. The presence of both open and closed conformations in the structures of R386K and E372T refutes the necessity for the interactive network in C-terminus movement. The data analysed throughout the project suggest simple mobility and thermal motion as the cause of the movement of the C-terminus. This work, in conjunction with kinetic data from the thesis of Helen Bell, presents structural data to characterise the role of binding residues within the active site of KMO as well as the mechanistic role of the C-terminus. It also highlights the importance of certain binding residues and countered the previously held hypotheses surrounding the significance of the C-terminus. The mechanistic role of the C-terminus therefore remains unclear and requires further study.

Doctor of Philosophy / Diagnostic Medicine/Pathobiology / Steven S. Dritz / The overall goal of this dissertation was to evaluate the effects of different amino acid (AA) levels on performance of pigs under commercial conditions. To reach this objective, a total of 12 experiments were conducted. For the sow research, 1,102 highly prolific sows were used to determine the effects of AA and energy intake during late gestation on piglet birth weight and reproductive performance of sows. Weight gain depended on the energy and AA intake levels while sows fed increased amount of energy had increased stillborn rate; however, there was no statistical differences due to energy intake in stillborn rate of gilts. The modest increase in individual piglet birth weight is due to energy rather than AA intake during late gestation. Pre-weaning mortality was reduced in piglets suckling from sows fed high AA diets during late gestation while subsequent reproductive performance was not affected. With recent advances in statistical computing capability, linear and non-linear mixed models were refined to estimate the AA ratio dose-response relationships. Then, 4 experiments using 2,420 wean-to-finish pigs were conducted to validate the methods for estimating the standardized ileal digestible (SID) AA to lysine (AA:Lys) ratio requirement. Subsequently, 7 experiments using 7,562 pigs were conducted to estimate the SID tryptophan (Trp) to Lys and Valine (Val) to Lys ratio requirements of wean-to-finish pigs. In 11- to 20-kg pigs, optimum SID Trp:Lys ranged from 16.6% for maximum mean G:F to 21.2% for ADG. In 30- to 125-kg pigs, optimum SID Trp:Lys ratio ranged from 16.9% for maximum mean G:F to 23.5% for ADG. However, 18% SID Trp:Lys captured 96 and 100% of the maximum mean ADG and G:F for finishing pigs, respectively. In 25- to 45-kg pigs, optimum SID Val:Lys ratio ranged from 72.3% for maximum mean G:F to 74.4% for ADG with 99% of the maximum mean ADG and G:F at approximately 69% and 65% SID Val:Lys ratio, respectively. In conclusion, optimum SID Trp:Lys and Val:Lys were consistently higher for ADG than G:F. This finding is critical for conducting economic evaluations and reference tables such as NRC (2012) should consider presenting requirement values for different response criteria.

Made available in DSpace on 2016-12-08T16:24:18Z (GMT). No. of bitstreams: 1 PGCA14MA140.pdf: 91454 bytes, checksum: d7f6d38c8c463559f1c52096a57acd9b (MD5) Previous issue date: 2014-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / We describe the epidemiological, clinical signs and lesions of four outbreaks of acute pulmonary edema and emphysema in cattle (EEPAB) in the states of Santa Catarina and Paraná and its experimental reproduction of disease. Spontaneous disease occurred after transfer of mature cattle grazing and dry for another young and flourishing. All affected cattle were cows of the Dutch and Swiss brown races. The main clinical signs were dyspnea , labored abdominal breathing with the neck extended and mouth open and some had subcutaneous emphysema , besides the decrease in milk production and slow recovery or death . Necropsy findings were restricted to the lung which had dark red color, not collapsed, glossy and hipercriptante with marked interlobular emphysema. Histological lesions in the lung consisted mainly of alveolar and interlobular emphysema interspersed with areas of congestion and edema, hyaline degeneration of the wall of alveoli and infiltration of macrophages and eosinophils , moderate , diffuse . The experimental reproduction of the disease was performed in a beef with administration of 0.7 mg / kg / BW of L - Tryptophan orally in a single dose. The animal died on the seventh day of the experiment. The clinical signs and lesions were similar to those observed in naturally occurring disease / Descrevem-se os dados epidemiológicos, sinais clínicos e lesões de quatro surtos da doença do edema e enfisema pulmonar agudo de bovinos (EEPAB) nos estados de Santa Catarina e Paraná e sua reprodução experimental. A doença espontânea ocorreu após transferência de bovinos de pastagem madura e seca para outra jovem e viçosa. Todos os bovinos afetados eram vacas das raças holandês e pardo suíço. Os principais sinais clínicos foram dispnéia, respiração abdominal dificultosa com o pescoço estendido e boca aberta e alguns apresentavam enfisema subcutâneo, além de queda na produção de leite e morte ou recuperação lenta. Os achados de necropsia foram restritos ao pulmão o qual tinha coloração vermelho escuro, não colabado, de aspecto brilhante e hipercriptante com enfisema interlobular acentuado. As lesões histológicas no pulmão consistiam principalmente de enfisema alveolar e interlobular intercalado por áreas de congestão e edema, degeneração hialina da parede de alvéolos e infiltrado de macrófagos e eosinófilos, moderado, difuso. A reprodução experimental da doença foi realizada em um bovino, com administração de 0,7mg/kg/PV de L-Triptofano por via oral em dose única. O animal morreu no sétimo dia de experimento. Os sinais clínicos e lesões foram idênticos aos observados na doença espontânea

Orientador: Elisabeth Criscuolo Urbinati / Banca: Antônio Fernando Gervásio Leonardo / Banca: Leonardo Sussumu Takahashi / Banca: Luciane Helena Gargaglioni Batalhão / Banca: José Augusto Senhorini / Resumo: Como estratégia para diminuir o alto índice de canibalismo na larvicultura do matrinxã, o uso do aminoácido essencial triptofano (Trp) parece promissor. O aminoácido é o precursor do neurotransmissor mono-amínico serotonina (5-hidroxitriptamina, 5-HT) e sua taxa de biossíntese no encéfalo são limitadas pela disponibilidade desse aminoácido, que parece ser uma das principais limitações para a síntese de 5-HT. A ingestão aumentada de triptofano eleva o nível do aminoácido no encéfalo, resultando em biossíntese aumentada de 5-HT no encéfalo de peixes. Esse por sua vez, é um neurotransmissor que media uma ampla variedade de comportamentos (agressão, medo e estresse em várias espécies, assim como na relação das interações sociais em muitos animais) por ação no sistema nervoso central e periférico. Assim, o presente estudo utilizou um aminoácido promotor da serotonina (triptofano), um inibidor seletivo da recaptação do neurotransmissor (fluoxetina) e um inibidor da síntese da serotonina (PCPA - paraclorofenilalanina), para verificar seu papel na mediação do comportamento agressivo e no desenvolvimento e sobrevivência larval de matrinxã no período de 1 a 12 dias após a eclosão. O triptofano foi fornecido juntamente com a ração, já a fluoxetina e a paraclorofenilalanina foram dissolvidos na água. Esse estudo foi realizado em duas condições de temperatura de cultivo, ou seja, com temperatura controlada em uma faixa de variação estreita (26-28°C) e com temperaturas variando de 19 a 31°C. Os resultados obtidos permitiram concluir que o uso do triptofano como suplemento na ração, pode ser uma alternativa viável na criação de matrinxã, pois aumentou o crescimento e a sobrevivência da prole, exceto na concentração mais alta (2,96g/100g ração). O uso de fluoxetina também aumentou a sobrevivência da prole, com redução do canibalismo, no ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of tryptophan as a strategy to reduce cannibalism in matrinxã hatchery seems promising. This amino acid is a precursor of the neurotransmitter serotonin (5-hidroxy-triptamine, 5-HT) whose biosynthesis rate in brain is limited by the amino acid availability. The increased intake of tryptophan by brain elevates the amino acid concentration in tissue resulting in higher serotonin production in fish. Serotonin is involved in the control of several types of behavior (aggression, fear, stress and social interaction in many animals) through effect in central and peripheral nervous systems. The present study evaluated the effect of a serotonin synthesis promoter (tryptophan), a selective inhibitor of serotonin reuptake (fluoxetine) and a selective inhibitor of serotonin synthesis (PCPA - paraclorofenilalanine) on the control of the aggressive behavior and on early development of matrinxã up to 12 days after hatching. Tryptophan was offered in the diet, and fluoxetine and PCPA in immersion solutions. The study was performed in two rearing temperatures, controlled temperature (26-28°C) and natural temperature without control (19 a 31°C). The results showed that tryptophan might be an alternative in matrinxã rearing since it promoted growth and enhanced survival of progeny, except in the highest concentration tested (2.96g/100g diet). Fluoxetine also enhanced larvae survival, with cannibalism reduction. The highest concentration (5000 ppb fluoxetine/L) reduced growth. The immunohistochemistry showed that the serotoninergig system is already developed in larvae and is similar to that of the juvenile fish of the same species. The temperature variation affected negatively the biological responses tested in this study, suggesting to be a stressor for fish. The PCPA did not show responses that could demonstrate a biological pattern / Doutor

Orientador: Carmen Silvia Passos Lima / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T11:41:22Z (GMT). No. of bitstreams: 1 Prestes_JulioCesar_M.pdf: 3086705 bytes, checksum: 732007aaed1cf1640977956c80551929 (MD5) Previous issue date: 2010 / Resumo: Segundo estimativas da Organização Mundial de Saúde, surgem a cada ano dez milhões de novos casos e cinco milhões de pessoas vão a óbito em decorrência do câncer. Apesar do aumento da especificidade de inúmeros novos quimioterápicos sobre as células tumorais, seus efeitos colaterais sobre células normais ainda representam um grande entrave no tratamento de portadores de câncer. Sinais e sintomas como alopecia, náuseas, vômitos, diarréia e adinamia tornam o tratamento muitas vezes inviável para muitos pacientes. Alguns suplementos nutricionais, como o selênio e a glutamina, foram descritos como eficientes para reduzir ou abolir os efeitos adversos da quimioterapia. O objetivo geral deste estudo é o de verificar se o suplemento nutricional TK3 (triptofano e timina), reduz as seguintes toxicidades: mucosite, diarréia, náuseas, vômitos, constipação intestinal, alopecta, dermatite, eritrodisestesia, anemia e astenia apresentadas por pacientes com câncer de mama metastático, câncer de cabeça e pescoço e câncer colorretal que receberam, em caráter adjuvante ou paliativo, os quimioterápicos cisplatina, doxorrubicina e 5-fluorouracil. Foi realizado um estudo duplo-cego, randomizado, prospectivo, com administrações diárias de TK3 (n=9) e placebo (n=11) e avaliações a cada três semanas, de acordo com as sessões de quimioterapia. Os efeitos colaterais foram avaliados por meio de um questionário específico e exame físico. A qualidade de vida foi avaliada por meio do questionário WHOQOL-bref. Não foi observada redução significativa da toxicidade da quimioterapia em pacientes que receberam TK3. Encontrou-se uma melhor qualidade de vida, no domínio psicológico na segunda visita, em pacientes com câncer de cabeça e pescoço que receberam TK3. / Abstract: According to estimates by the World Health Organization, appear each year ten million new cases and five million people will die due to cancer. Despite the increased specificity of several new chemotherapeutic agents on the tumor cells, their side effects on normal cells still represent a major obstacle in treating cancer patients. Signs and symptoms such as alopecia, nausea, vomiting, diarrhea and malaise often make treatment unaffordable for many patients. Some nutritional supplements such as selenium and glutamine, were described as effective to reduce or abolish the adverse effects of chemotherapy. The aim of this study is to determine whether the nutritional supplement TK3 (tryptophan and thymine), reduces the following toxicities: mucositis, diarrhea, nausea, vomiting, constipation, alopecia, dermatitis, erythrodysesthesia, anemia and asthenia presented by cancer patients metastatic breast cancer, head and neck and colorectal cancer who received adjuvant or palliative in nature, the chemotherapy drugs cisplatin, doxorubicin and 5-fluorouracil. We conducted a randomized double-blind, randomized, prospective, with daily doses of TK3 (n=9) and placebo (n=11) and evaluations every three weeks, according to the chemotherapy sessions. Side effects were assessed using a questionnaire and physical examination. Quality of life was assessed by the WHOQOL-bref. There was no significant reduction of the toxicity of chemotherapy in patients who received TK3. We found a better quality of life in the psychological field in the second visit, in patients with head and neck cancer who received TK3. / Mestrado / Clinica Medica / Mestre em Clinica Medica

Orientadores: João Ernesto de Carvalho, Ana Lucia Tasca Gois Ruiz / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T11:07:54Z (GMT). No. of bitstreams: 1 Monteiro_KarinMaia_M.pdf: 2254231 bytes, checksum: 20b2ee4f5d5aaf0109e94552d1fec08d (MD5) Previous issue date: 2006 / Resumo: O complemento nutricional ¿TK3¿ foi inicialmente estudado por um bioquímico alemão, Sr. Friedrich Lavitcshka, que desenvolveu uma formulação em 1969, inicialmente líquida, constituída basicamente pela associação de triptofano e timina. Seus estudos em modelos experimentais de tumores de pele induzidos por alcatrão em ratos não foram publicados. Entretanto, seus resultados, bem como relatos de casos provenientes do uso informal de tal suplementação, apontavam para efeitos benéficos superiores ao de um mero complemento nutricional ou ¿fortificante¿, como se costumava denominar tal categoria de substâncias. A avaliação toxicológica deste complemento, como parte inicial deste estudo pré-clínico, revelou: 1. Estudos ¿in vitro¿: atividade não citotóxica de ¿TK3¿ em cultura de células humanas normais ¿ fibroblastos ¿ e tumorais; 2. Estudos agudos ¿in vivo¿: a administração aguda de ¿TK3¿ (5 g/Kg, p.o. e 2g/Kg, i.p.) em ratos Wistar não produziu quaisquer sinais clínicos de toxicidade. A análise macroscópica dos órgãos destes animais experimentais não revelou quaisquer alterações sugestivas de toxicidade; 3. Estudos sub-crônicos ¿in vivo¿: a administração doses-repetidas 90 dias de ¿TK3¿ (100 mg/Kg, 300 mg/Kg e 1000 mg/Kg, p.o.) em ratos Wistar de ambos os sexos igualmente não revelou quaisquer sinais de toxicidade, confirmados pela análise macroscópica dos órgãos. Finalmente, as análises hematológicas e bioquímicas não identificaram alterações indicativas de toxicidade. A triagem farmacológica deste complemento revelou: 1. Em modelo de úlcera induzida por etanol em ratos , o tratamento com ¿TK3¿ (1000 mg/Kg, p.o.) reduziu o índice de lesão ulcerativa (86.2%). Esta atividade antiulcerogênica foi superior àquela apresentada pela administração dos componentes isoladamente (triptofano: 400mg/Kg [61.5%] e timina: 400 mg/Kg [+30.8%]); 2. Na avaliação da atividade antisecretora, em modelo de ligadura de piloro por 4 horas, a administração intraduodenal de ¿TK3¿ (1000 mg/Kg, p.o.) não reduziu o volume, nem a acidez total e nem aumentou o pH da secreção ácida gástrica basal; 3. Na triagem dos mecanismos de citoproteção gástrica, o pré-tratamento de ratos com indometacina (5 mg/Kg, p.o.) não inibiu o efeito protetor do ¿TK3¿ (1000 mg/Kg, p.o.) nas lesões induzidas por etanol; 4. O pré-tratamento de ratos com L-NAME (5 mg/Kg, i.v.) não inibiu o efeito protetor do ¿TK3¿ (1000 mg/Kg, p.o.) nas lesões induzidas por etanol; 5. Finalmente, o pré-tratamento de ratos com NEM (10 mg/Kg, s.c.) foi capaz de inibir em 50% o efeito protetor do ¿TK3¿ (1000 mg/Kg, p.o.) nas lesões induzidas por etanol, sugerindo a participação de compostos contendo grupos sulfidrila no mecanismo de ação do referido complemento / Abstract: The nutritional supplement ¿TK3¿ was originally studied by a German biochemist, Mr. Friedrich Lavitcshka, who first associated L-tryptophan and thymine, in a liquid formula, in 1969. His early studies using experimental tar-induced skin cancer models were not published even though they seemed to confirm the benefits reported by informal use of such association at that time. These reports suggested some pharmacological effects beyond the nutritional one. The toxicological evaluation of this new association of substances, as part of its pre-clinical trial, revealed: 1. ¿In vitro¿ studies: no citotoxicity activity of ¿TK3¿ on normal (fibroblasts) and cancer human cells; 2. ¿In vivo¿ acute studies: acute administration of ¿TK3¿ (5 g/Kg, p.o. and 2g/Kg, i.p.) in Wistar rats revealed no signs of systemic toxicity or impending death; 3. ¿in vivo¿ sub-chronic studies: 90 day repeated-dose study of ¿TK3¿ (100 mg/Kg, 300 mg/Kg and 1000 mg/Kg, p.o.) in Wistar rats of both sexes revealed no clinical signs of toxicity, confirmed by both histopathological and biochemical analysis. The pharmacological screening of this nutritional supplement revealed: 1. In the ethanol-induced gastric ulcer experimental model in rats, the treatment with ¿TK3¿ (1000mg/Kg, p.o.) reduced the ulcerative ulcer index (86.2%). This antiulcerogenic activity was superior to the ones presented by the isolated components administrated individually (L-tryptophan: 400mg/Kg [61.5%] and thymine: 400mg/Kg [+30.8%]); 2. In the evaluation of gastric secretion through pyloric ligation model in rats, the treatment with ¿TK3¿ (1000 mg/Kg, i.d.) had no significant effect over the gastric juice volume, nor the pH or hydrogenionic concentration of gastric content; 3. In the evaluation of possible gastric cytoprotective mechanisms of action, pre-treatment of experimental rats with indomethacin (5 mg/Kg, p.o.) did not inhibit the antiulcerogenic effect of ¿TK3¿ (1000 mg/Kg, p.o.) in the ethanol induced ulcer model; 4. Similarly, pre-treatment of experimental rats with L-NAME (5 mg/Kg, i.v.) did not inhibit the antiulcerogenic effect of ¿TK3¿ (1000 mg/Kg, p.o.) in the ethanol induced ulcer model; 5. Finally, pre-treatment of experimental rats with NEM (10 mg/Kg, s.c.) decreased 50% of the antiulcerogenic effect of ¿TK3¿ (1000 mg/Kg, p.o.) in the ethanol induced ulcer model, suggesting the participation of endogenous non-protein SH-containing compounds. However, as this reduction was not complete, there seems to be more aspects involved in this mechanism of action / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

A via das Quinureninas (Via Quin) representa a principal via catabólica do metabolismo do triptofano (Trp) e é essencial para diversos processos fisiológicos. No fígado, o Trp é catalisado por triptofano 2,3-dioxigenase (TDO) quinurenina (Quin). A mesma reação também pode ser catalisada pela enzima indolamina 2,3-dioxigenase (IDO), produzida por células imunológicas. Em alguns processos patológicos, há um aumento do consumo de Trp pela Via Quin, que gera compostos que estão relacionados ao processo de imunotolerância. No presente estudo, foram selecionados dois modelos que mimetizam situações associadas às alterações da resposta imunológica: a restrição de sono e a obesidade. A partir do conhecimento das alterações na resposta imune nessas condições, geramos a hipótese de que parte do mecanismo se dê a partir da indução do catabolismo de Trp pela via Quin. Desse modo, foram investigadas as concentrações séricas e hepáticas de Trp nesses modelos experimentais, modelos esses que foram utilizados em outros projetos do nosso grupo de pesquisa. Não houve diferença significativa na concentração de Quin sérica e hepática entre os camundongos C57BL/6J restritos de sono (3 hs/15 dias), privação de sono paradoxal (72 hs) e período rebote (24 hs). A razão Quin/Trp também não diferiu entre os grupos RS e controle. Igualmente não houve diferenças estatísticas na concentração de Quin plasmática nos modelos privação de sono paradoxal e período rebote realizados em ratos Wistar. O mesmo foi observado em camundongos Swiss e camundongos C57BL/6J submetidos a protocolos experimentais de obesidade: ração hiperlipídica (21 dias) e de síndrome metabólica (20 semanas de ração hiperlipídica). Tais resultados sugerem que as alterações na resposta imunológica nesses quadros não estão associadas ao catabolismo de Trp. / The Kynurenine pathway (Kyn pathway) is the major catabolic pathway of tryptophan metabolism (Trp) and it is essential for many physiological processes. In the liver, Trp is catalyzed by tryptophan 2,3-dioxygenase (TDO), producing kynurenine (Kyn). The same reaction can also be catalyzed by the enzyme indoleamine 2,3-dioxygenase (IDO), produced by immune cells. In some pathological conditions, there is a high Trp consumption by Kyn pathway, that generate compounds related to immune tolerance. In this study, we chose two models strongly associated with changes in the immune response: sleep restriction and obesity. From the knowledge that there are immune response alterations in those conditions, we generated the hypotesis that in part, those alterations are correlated with induction the Trp catabolism by Kyn pathway. Thus, serum and liver concentrations of Trp and Kyn were investigated in these experimental models that have been used in other projects of our research group. There was no significant difference in concentration of Kyn in serum and liver among mice C57BL/6J induced to restricted sleep (3 hours / 15 days), paradoxical sleep deprivation (72 hours) and rebound period (24 hours). The Kyn/Trp ratio did not differ between control group and RS group. Also there were no statistical differences in plasma concentration of Kyn in paradoxical sleep deprivation and rebound period models performed in rats Wistar. The same profile was also observed in Swiss e C57BL/6J mice subjected to experimental obesity protocols: fat diet (21 days) and metabolic syndrome (20 weeks of fat diet). These results suggest that changes in the immune response in the conditions tested above are not associated with Trp catabolism.

O triptofano (Trp) é essencial para muitos processos fisiológicos e seu metabolismo apresenta-se alterado em doenças como no câncer. O Trp é degradado por três vias: via das quinureninas; via serotonérgica e via das triptaminas. A primeira está envolvida com a tolerância e o imune escape de células tumorais, já a segunda leva a produção de serotonina e melatonina, com uma diversidade de funções biológicas e que possuem atividades antitumorais reconhecidas. A terceira rota é a menos estudada e a função dos compostos sintetizados ainda é desconhecida. Trabalhos do grupo mostram que a via das triptaminas é ativa em melanomas e que triptamina (TRY) e dimetiltriptamina (DMT), metabólitos produzidos por esta via, também possuem atividade antitumoral. Nosso objetivo é avançar sobre a compreensão de como esta via afeta o metabolismo e crescimento tumoral. Para tanto, estudamos mais detalhadamente a via em linhagens de melanoma. A adição de TRY e DMT nas culturas modificou a produção de compostos das outras rotas de metabolização do Trp. Além disso, TRY e DMT afetaram a invasividade das células tumorais e TRY aumentou a atividade citotóxica de células mononucleares frente a melanomas. Observamos que apesar dos efeitos biológicos da via das quinureninas estar amplamente relacionado a ligação dos metabólitos no receptor de aril hidrocarbonetos, para a via das triptaminas o receptor parece não estar associado com a atividade antitumoral descrita. Nossos resultados apontam para a importância da via das triptaminas na dinâmica tumoral e a necessidade de estudos mais amplos relacionados ao metabolismo do Trp. / Tryptophan (Trp) is essential for many physiological processes and its metabolism is modified in several diseases such as in cancer. TRP is broken down into three pathways: kynurenine path, serotonergic path and tryptamine path. The first is involved in tolerance and immune escape of cancer cells, while the second leads to the production of serotonin and melatonin, which have a variety of biological functions and recognized antitumor activity. The third route is the least studied and the biological function of the synthesized compounds is still unknown. Our group shows that tryptamine path is active in melanomas and tryptamine (TRY) and dimethyltryptamine (DMT), metabolites produced by this route, also have antitumor activity. Our goal is make progress on understanding how tryptamine route affects metabolism and tumor growth. Therefore, we studied in detail this pathway in melanoma cell lines. The addition of TRY and DMT into the cultures leds the production of metabolites of other Trp routes. Moreover, TRY and DMT affect the invasiveness of tumor cells and TRY increased antitumor activity of the immune system against melanomas. We observed that despite biological effects of kynurenine path be largely related to metabolites binding aryl hydrocarbon receptor, for tryptamine pathway the receptor seems not to be associated with the described antitumor activity. Our results point the importance of tryptamine pathway in tumor dynamics and the need for broader studies related to Trp metabolism.

Neste trabalho, são estudados dois dipeptídeos complexados com cobre, Triptofil-Glicina (trp-Gly) e Glicil-Triptofano (Gly-Trp). Focalizamos a interação com o metal de transição, mudanças conformacionais, papel do resíduo pesado de triptofano na simetria dos complexos formados, e explicamos as propriedades espectroscópicas atípicas que observamos no Gly-Trp: Cu+2 em pH´s altos semelhantes aquelas apresentadas em uma espécie de proteínas naturalmente complexadas as proteínas azuis. Para atingirmos tais objetivos, lançamos mão das técnicas de RPE, Dicroísmo Circular (CD) e Absorção ótica, pelas suas complementaridades. Com os dados experimentais, propusemos dois modelos para os complexos com suas funções de onda. Para Trp-Gly: Cu+2, nos pH´s 9,1 e 13,2 e para o Gly-Trp: Cu+2 no pH= 9,1 propomos um modelo que chamamos de covalente. Que consiste basicamente em considerar que os orbitais do íon Cu+2 e seus ligantes, se encontram em simetria quadrado planar (D4h) e que cada ligante no caso nosso, dois oxigênios e dois nitrogênios, tem disponíveis seus orbitais 2s, 2px, 2py, 2pz para a formação dos orbitais moleculares com os orbitais 3d do cobre. As funções assim construídas, dependem de coeficientes que nos dão informações sobre o grau de covalência. O método é semi-empírico. As expressões teóricas dos parâmetros RPE dependem destes parâmetros de covalência e das energias de transição obtidas por absorção ótica no visível. Pudemos então com os valores experimentais das componentes dos tensores g e A obtermos os valores numéricos dos parâmetros de covalência. Para o complexo Gly-Trp: Cu+2, no pH=13,2, propusemos o modele de mistura de orbitais, um modele não covalente que consiste em considerarmos as funções de onda dos estados excitados 4s e 4p do cobre misturadas com as dos orbitais 3d do mesmo íon, para explicar as propriedades espectroscópicas pouco comuns, que se deslocam na direção daquelas obtidas nas proteínas azuis. Conhecendo os valores experimentais das componentes dos tensores g e A, da força de oscilador obtida dos espectros óticos e da força rotacional obtida dos espectros CD, e das auto-funções compatíveis com o modelo, determinamos numericamente os coeficientes de hibridização (mistura). Os parâmetros experimentais, foram determinados através de simulações espectrais, utilizando programas desenvolvidos para este fim. Através deste trabalho, pudemos enfim, verificar a influência do resíduo pesado de triptofano na estereoquímica dos complexos formados, mudanças de simetria, o caráter das ligações, e ate que ponto as propriedades do dipeptídeo Gly- Trp: Cu2+ em pH alto, são semelhantes às proteínas azuis / In this work we studied two dipeptide-copper complexes, being Triptofil-Glycine (Trp-Gly) and Glycil-Triptophan (Gly-Trp). We focused our interest on the interaction with the transition metal, conformational changes and the role that the heavy residue of the Triptophan plays in the symmetry of the complex. Moreover we explain the unusual spectroscopic properties that we observed with Gly-Trp: Cu+2 in high pH solutions similar to that of the so called blue proteins. For these purposes we used such complementary techniques as EPR, Circular Diochrism and Optical Absorption Spectroscopy. Based on our experimental results we proposed two models for the complexes and their wave functions. For the TRP-Gly: Cu+2 in pH of 9.1 and 13.2 and for Gly-Trp: Cu+2 with pH=9.1, we use a model which we call covalent. It consists basically of the consideration that the electronic orbital of the ion Cu+2 and its ligands are in square planar symmetry and that each of the four ligand atoms, in our case two oxygen and two nitrogen atoms, has available 2s, 2px, 2py, 2pz for the formation of molecular orbital with the copper 3d orbital. The wave functions thus constructed depend on coefficients (parameters) that give information on the degree of the covalent character of the bond (complex). The method is semi-empirical. The theoretical expressions for the EPR parameters depend on these coefficients and the transition energies obtained by absorption spectroscopy (in the visible). With the experimental values of the g and A tensors we can therefore obtain numerical values for the covalent coefficients. For the complex Gly-Trp: Cu+2 with pH=13.2 we proposed a model of mixing orbital, a non covalent model that consists of the assumption that the wave functions of the excited states 4s and 4p of the copper mix with the 3d orbital of the same ion. With this model it was possible to explain the uncommon spectroscopic properties that are similar to these of the blue proteins. Knowing the experimental values of the components of the tensors g and A, the oscillator strength obtained by the optical spectra, the rotational strength obtained by the CD spectra and the eigen-functions compatible with the model, we numerically determined the coefficients of hybridization. The experimental parameters were determined by spectral simulations using programs that we developed especially (specifically) for this purpose. Trough this work it was possible to verify the influence of Triptophan on the stereochemistry (stereo chemical behavior) of the formed complexes, on symmetry changes, covalent character and up to which point the properties of the dipeptide Gly-Trp: Cu+2 in high pH are similar to that of the blue protein