The development of fatigue has been an area of interest to athletes and scientists alike for many years. Often, particularly during prolonged exercise in the heat, there is no obvious peripheral reason for fatigue and the central nervous system is cited as the source. The mechanisms and potential manipulations of this fatigue remain largely unclear. Chapters Three and Four attempted to reduce the transport of the serotonin precursor tryptophan into the brain in order to reduce or delay serotonin synthesis and therefore increase exercise capacity. In Chapter Three branched-chain amino acid drinks were fed before and during prolonged cycling to exhaustion in the heat on two occasions and control drinks were fed on two other occasions. There was no effect of the branched-chain amino acids on exercise capacity and the intra-individual variability in seven of the eight participants was small. One participant did appear to cycle for longer on the branched-chain amino acid trials compared to the control trials. In Chapter Four a 104 g bolus of amino acids, designed to deplete plasma tryptophan concentration, was fed seven hours before a prolonged cycle to exhaustion in the heat. There was no difference in exercise capacity between the tryptophan depletion trial and the control trial in which tryptophan was also ingested. These findings suggest that the delivery of tryptophan to the central nervous system is not the only factor influencing the onset of fatigue. The investigation undertaken in Chapter Five looked at the serotonin transporter density on the blood platelets of current and retired international level athletes competing in either endurance or sprint running events and a sedentary control group. Using the platelet as an accessible and reliable model for the serotonergic neuron, the maximum number of binding sites was assessed using the radio-labelled serotonin reuptake inhibitor [3H]Paroxetine. Those currently training for endurance events had a greater number of binding sites than any of the other groups. This supports previous findings and suggests that endurance training can increase the number of serotonin transporters on blood platelet membranes. During resting heat exposure in Chapter Six, the application of a 1 % menthol solution to the skin of the forearms, back and forehead elicited a warming sensation in some individuals and a cooling sensation in other individuals, but never any change in skin or core temperature nor skin blood flow. A small proportion of individuals did not perceive any change in skin thermal sensation. Chapter Seven applied these findings to a pre-loaded twenty minute exercise performance test in the heat. It was hypothesised that those who perceived a warming effect may perform worse when a menthol solution was applied compared to a control solution and conversely, those who perceived a cooling sensation may perform better with a menthol solution than with a control solution. There was no difference in exercise performance between those who felt a warming sensation and those who felt a cooling sensation. Those who felt a warming sensation felt significantly warmer on the menthol trial than the control trial but this did not affect their performance. However, those who reported a cooling sensation tended to feel cooler on the menthol trial than the control trial, and there was a tendency for an improvement in performance on the menthol trial compared to the control trial. Due to the experimental protocols adopted in this thesis it was possible to assess the reliability of an exercise capacity test compared to an exercise performance test. Chapter Three showed a coefficient of variation of 11.0 ± 11.2 % and Chapter Four showed a 11.5 ± 12.4 % variability for exercise capacity tests. Chapter Seven showed a coefficient of variation in a pre-loaded time-trial exercise performance test of 3.9 ± 9.6 % suggesting that an exercise performance test may be more reliable than an exercise capacity test. However, the aims of an investigation are still likely to be the main factor influencing the choice of protocol. It seems likely that no single mechanism will be responsible for the cessation of exercise. The investigations undertaken in this thesis also highlight many avenues for future exploration.

<p>Nature is challenged to move charge efficiently over many length scales. From sub-nm to μm distances, electron-transfer proteins orchestrate energy conversion, storage, and release both inside and outside the cell. Uncovering the detailed mechanisms of biological electron-transfer reactions, which are often coupled to bond-breaking and bond-making events, is essential to designing durable, artificial energy conversion systems that mimic the specificity and efficiency of their natural counterparts. Here, we use theoretical modeling of long-distance charge hopping (Chapter 3), synthetic donor-bridge-acceptor molecules (Chapters 4, 5, and 6), and de novo protein design (Chapters 5 and 6) to investigate general principles that govern light-driven and electrochemically driven electron-transfer reactions in biology. We show that fast, μm-distance charge hopping along bacterial nanowires requires closely packed charge carriers with low reorganization energies (Chapter 3); singlet excited-state electronic polarization of supermolecular electron donors can attenuate intersystem crossing yields to lower-energy, oppositely polarized, donor triplet states (Chapter 4); the effective static dielectric constant of a small (~100 residue) de novo designed 4-helical protein bundle can change upon phototriggering an electron transfer event in the protein interior, providing a means to slow the charge-recombination reaction (Chapter 5); and a tightly-packed de novo designed 4-helix protein bundle can drastically alter charge-transfer driving forces of photo-induced amino acid radical formation in the bundle interior, effectively turning off a light-driven oxidation reaction that occurs in organic solvent (Chapter 6). This work leverages unique insights gleaned from proteins designed from scratch that bind synthetic donor-bridge-acceptor molecules that can also be studied in organic solvents, opening new avenues of exploration into the factors critical for protein control of charge flow in biology.</p> / Dissertation

O melanoma é composto por células malignas e também por um estroma de sustentação que inclui fibroblastos, células imunológicas, endoteliais, matriz extracelular, dentre outros fatores. Assim, os tumores não são entidades independentes, eles interagem ativamente com o microambiente adjacente de forma bidirecional através de sinais moleculares que modulam o fenótipo maligno. Um dos sinais bioquímicos para desenvolvimento desse fenótipo se dá pelo catabolismo de Trp pela via das quinureninas, que gera compostos com diversas atividades biológicas, que no tumor estão envolvidas com tolerância e imunoescape e, logo, com prognóstico ruim para os pacientes. Até o presente momento apenas o consumo de Trp e a formação de um único metabólito, a quinurenina (KYN), tem sido associada a malignidade dos melanomas. A fim de ampliar e elucidar os mecanismos bioquímicos do metabolismo desse aminoácido em melanomas, estudamos mais de quinze compostos de todas as rotas catabólicas de Trp em células da pele, células imunológicas, linhagens tumorais e amostras clínicas de melanoma. De forma inédita pudemos observar que as células da pele tem maior habilidade de sintetizar KYN quando comparadas às linhagens tumorais, demonstrando que o catabolismo de Trp peritumoral pode ser responsável pelos fenômenos de imunotolerância e escape. Além disso, o metabolismo de Trp pode estar envolvido nos mecanismos de homeostasia da pele, já que especificamente essas células produzem compostos com atividade biológica nesse órgão. As células imunológicas possuem um perfil metabólico completamente diferente umas das outras: monócitos, macrófagos e dendríticas possuem maior ativação da via KYN enquanto linfócitos e neutrófilos possuem maior indução da rota que gera serotonina e melatonina. Mesmo nos diferentes fenótipos de macrófagos, M1 e M2a, foram observadas marcações especificas de metabolismo, que podem estar relacionadas às atividades anti- ou pró-tumoral dessas células no microambiente. Em amostras clínicas, apesar da principal diferença entre nevos e melanomas ser a concentração de KYN, diversas outras alterações no metabolismo de tiptofano foram observadas, o que mostra a complexa magnitude deste metabolismo na fisiopatologia da pele / Melanoma is composed of malignant cells and also by a stromal support that includes fibroblasts, immune cells, endothelial cells, extracellular matrix, among other factors. Thus, tumors are not separate entities; they actively interact with the surrounding microenvironment bi-directionally through molecular signals that modulate the malignant phenotype. One of biochemical signals for the development of this phenotype occurs by Trp catabolism through kynurenine pathway, that generates compounds with diverse biological activities, which in tumors are involved with tolerance and imunoescape and therefore with poor prognosis for patients. To date only the consumption of Trp and formation of a single metabolite, kynurenine (KYN), has been associated with malignant melanomas. In order to enlarge and clarify the biochemical mechanisms of this amino acid metabolism in melanomas, we have studied more than fifteen compounds of all catabolic routes of Trp in skin cells, immune cells, tumor cell lines and clinical samples of melanoma. In an unique way we could observe that the skin cells has superior ability to synthesize KYN when compared to tumor cell lines, demonstrating that the peritumoral catabolism of Trp may be responsible for the phenomena of immune tolerance and escape. Furthermore, the Trp metabolism may be involved in skin homeostasis mechanisms, since these cells produce specific compounds with biological activity in this organ. The immune cells have a completely different metabolic profile among them: monocytes, macrophages and dendritic cells have greater KYN pathway activation, and lymphocytes and neutrophils possess greater induction of the route that generates serotonin and melatonin. Even in different macrophages phenotypes, M1 and M2a, we observed specific metabolic marks, which may be related to the anti- or pro-tumoral activity of these cells in the tumor microenvironment. In clinical samples, although the main difference between nevi and melanomas is the concentration of KYN, a range of other changes in Trp metabolism were observed, which shows the complex magnitude of this metabolism in the skin pathophysiology

Introdução: existe evidencia de que o sabor umami detecta os alimentos ricos em proteínas e aminoácidos, o que presumivelmente interfere na ingestão destes alimentos. Há experimentos publicados que relacionam quadros de desnutrição ou alimentação deficiente de determinados aminoácidos, com o consumo voluntário de soluções ou ração contendo tais aminoácidos. Objetivos: os objetivos deste trabalho foram: a caracterização das preferências gustativas para diferentes aminoácidos no rato normal e o efeito da desnutrição protéica sobre essas preferências. Metodologia: o estudo compreendeu duas fases distintas: na primeira foram feitas comparações de consumo de soluções de aminoácidos isolados (10 gramas por litro de água) em um período curto (5 dias). As comparações foram realizadas em 4 grupos distintos, e cada grupo foi composto de 20 animais: 10 controles e 10 desnutridos. A desnutrição foi induzida pelo oferecimento de ração hipoprotéica (5% de caseína na primeira fase e 7,5% de caseína na segunda fase). As soluções de aminoácidos comparadas em cada grupo foram: glutamato e glicina, glutamato e valina, triptofano e glicina e triptofano e valina, todas na concentração de 10g/ L. Na segunda fase foram realizadas comparações de consumo de soluções contendo glutamato mais um aminoácido não essencial (glicina) e glutamato mais um aminoácido essencial (lisina ou triptofano) por um período de 42 dias. Esta segunda fase foi composta de 3 grupos de animais, sendo um formado por 10 animais controles e 10 desnutridos que não receberam soluções, outro grupo com a mesma divisão de animais, mas que receberam soluções de glutamato mais glicina e glutamato mais lisina, e o último grupo recebeu soluções de glutamato mais glicina e glutamato mais triptofano. Resultados, discussão e conclusão: observou-se, tanto na fase 1 como na fase 2, que: a glicina e o glutamato em solução são muito atrativos; a de lisina é atrativa 4 mas em menor intensidade do que as anteriores; a de valina é pouco atraente; o consumo da solução de triptofano foi tão pequena que esta é possivelmente aversiva. Os resultados da fase 1 indicaram que o consumo de soluções de glutamato e de valina de animais desnutridos foi significativamente maior do que o de animais controles. Entretanto, no subgrupo de ratos desnutridos, o consumo da solução de glutamato correlacionou-se positivamente com o peso do animal. Na fase 2 os animais desnutridos, nas duas últimas semanas experimentais, mas não nas anteriores, apresentaram consumo significativamente maior de lisina e triptofano; a comparação do consumo de solução de glicina foi impossibilitada pelo consumo excessivo e esgotamento dos frascos. Também se constatou que os animais da segunda fase apresentaram esteatose hepática. Entre os desnutridos, o consumo de solução de lisina foi inversamente correlacionado com o peso corporal / Umami is the taste quality associated with several amino acids, especially the amino acid L-glutamate, and evidence exists that umami indicates the presence of amino acids, peptides and related structures in foodstuffs, which may bear relevant nutritional implications. The objectives of the study were to establish the normal rat preferences among a set of amino acids (glycine, glutamate, lysine, valine and tryptophan), and determine wether the rat protein malnutrition modifies the preferences and/or the avidity for these amino acids. In a initial set of experiments, bottles containing 50 ml of the amino acid solutions whose palatability were to be compared were presented to each animal for a 5-day period, and volumes consumed during the last 3 days were measured, and the bottles were refilled to their original levels. The left-right positions of the bottles were reversed every day. At the termination of the testing periods, the volumes of test solutions remaining in each bottle were measured, and the consumed volumes calculated. In a second set of experiments, solutions of glutamate plus glycine, glutamate plus lysine or glutamate plus tryptophan were presented to the animals for 8 weeks; after that , for 2 weeks, the glutamate was removed so that solutions of glycine, lysine or tryptophan only were presented to the animals. The volumes remaining in the bottles were measured and the volumes consumed calculated at termination of each testing period. On the basis of the ratios of volume of solutions consumed/ body weight the avidity for the amino acid were inferred. Protein malnutrition was induced by a diet with 5% -7.5 % casein as the only protein source; malnutrition was consistently induced as demonstrated by severe reduction in weight gain and increase in liver fat content. Normal rats demonstrated great avidities for glycine and glutamate, moderate avidity for lysine, low avidity for valine and no avidity for tryptophan. Rats with protein malnutrition a similar profile of avidities, however they significantly grater avidities for 6 glutamate, lysine, valine, and tryptophan, but not for glycine than normal rats. Among the rats with with protein malnutrition, greater the body weight, the greater the glutamate solution comsumed/body weight and the lower the body weight, the greater the lysine solution consumed /body weight. Conclusions: Avidity for amino acid solutions is widely variable, and protein malnutrition enhances the avidity for glutamate, valine, lysine, and tryptophan solutions but not for glycine solution

O tumor de Ehrlich (TE) foi descrito inicialmente, como adenocarcinoma mamário de camundongos, desenvolvendo-se na forma ascítica ou sólida a partir de sucessivos transplantes no peritônio ou tecido subcutâneo destes animais. O tumor ascítico de Ehrlich (TAE) tem sido utilizado como um tumor transplantável para o desenvolvimento de pesquisas relacionadas à oncologia. Estudos tem demonstrado que o desenvolvimento de TAE resulta no estímulo de células citotóxicas, como os linfócitos T e células Natural Killer (NK), mediadas principalmente por macrófagos. Os macrófagos e as células dendríticas (DCs), podem induzir a síntese da enzima indoleamina 2,3 dioxigenase (IDO) em tecidos tumorais, via ligação das moléculas co-estimuladoras B7-1 e B7-2, presentes nestas células, com a molécula CTLA-4 (antígeno 4 associado a linfócitos T citotóxicos), presente em linfócitos T reguladores CD4+ CD25+. A IDO é uma enzima que degrada o aminoácido essencial triptofano, processo que além de levar à privação do mesmo no microambiente celular, gera metabólitos que impedem a ativação e proliferação de linfócitos T e, consequentemente, mecanismos a eles associados como os de rejeição podem ser seriamente comprometidos. Mediante o exposto e pela presença de células imunológicas que expressam IDO no microambiente do tumor ascítico de Ehrlich, este trabalho teve como objetivo verificar a expressão da IDO após o bloqueio da interação B7/CTLA-4 por meio da citometria de fluxo. De acordo com as análises realizadas, os resultados demostraram que houve redução de 4,9% para 2,53% na expressão da enzima IDO. Em face dos resultados, parece plausível sugerir que o bloqueio desta via de ligação foi eficaz na redução dos níveis de atividade da IDO, o que poderia restaurar a capacidade de resposta dos linfócitos T contra as células tumorais. Nesta perspectiva sobre a IDO como mediadora no controle do escape imune feito pelas células tumorais, tais resultados podem colaborar para modulação desta enzima no microambiente tumoral. / The Ehrlich tumor (TE) was first described, as breast adenocarcinoma of mice. The TE develops in ascitic or solid form from successive transplantations in the peritoneum or subcutaneous tissue of these animals. The Ehrlich ascites tumor (TAE) has been used as a transplantable tumor for the development of research related to oncology. Studies have shown that the development of TAE results in stimulation of cytotoxic cells, such as T lymphocytes and Natural Killer cells (NK), mediated mainly by macrophages. Macrophages and dendritic cells (DCs) in tumor tissues, via co-stimulation of molecules B7-1 and B7-2, present in these cells, toghether with the CTLA-4 (Cytotoxic T-Lymphocyte-Associated antigen 4) molecule, present on regulatory T CD4 + CD25 + lymphocytes , may induce the synthesis of indoleamine 2, 3 dioxygenase (IDO). IDO is an enzyme that catabolizes the essential amino acid tryptophan, impairing activation and proliferation of T lymphocytes and consequently, compromising mechanisms associated with them such as rejection. Considering the presence of immune cells in the tumor microenvironment Ehrlich ascites that express IDO, this study aimed to verify the expression of IDO after the blockade of interaction B7/CTLA-4 by flow cytometry. Results demonstrated that there was a reduction of 4.9% to 2.53%in the expression of IDO. Given the results, it seems plausible to suggest that blocking this binding via was effective in reducing the levels of expression of IDO, which could restore the responsiveness of T cells against tumor cells. In this perspective on the IDO as a mediator in the control of immune escape made by tumor cells, these results may collaborate for modulation of this enzyme in the microenvironment.

Le tryptophane, un acide aminé essentiel, est soit utilisé pour la synthèse protéique et la synthèse de sérotonine, soit dégradé en plusieurs métabolites appelés collectivement les kynurénines. L’expression et l’activité des enzymes de la voie des kynurénines sont stimulées par l’inflammation. La synthèse des kynurénines est donc susceptible d’être augmentée chez les individus obèses. En effet, l’obésité est caractérisée par une inflammation chronique à bas bruit du tissu adipeux, reflétée par l’augmentation de facteurs inflammatoires circulants qui contribuent à l’apparition de l’insulinorésistance et du diabète de type 2. Plusieurs métabolites de la voie des kynurénines pourraient être des facteurs de risque pour le développement de l’insulinorésistance. La chirurgie bariatrique est actuellement le traitement le plus efficace pour l’obésité sévère, elle permet une perte de poids significative ainsi qu’une diminution des facteurs inflammatoires circulantes et une amélioration de l’insulinorésistance et du diabète. Il a été démontré que l’expression d’IDO1, la première enzyme de la voie des kynurénines, est plus élevée dans le tissu adipeux des individus obèses. Le ratio kynurénine sur tryptophane, qui reflète l’activité D’IDO1, est également augmenté chez les individus obèses.Notre objectif a été de caractériser l’expression des enzymes de la voie des kynurénines dans le tissu adipeux et d’évaluer les concentrations des kynurénines dans les sérums de patientes obèses pour rechercher si certains de ces facteurs pouvaient être reliés à l’apparition du diabète. Ces études ont été réalisées dans une cohorte de femmes obèses normoglycémiques et diabétiques. Puis dans un second temps nous avons étudié les conséquences de la perte de poids induite par la chirurgie bariatrique sur les concentrations circulantes des kynurénines et évalué si les variations des concentrations des kynurénines pourraient expliquer en partie l’amélioration du diabète observée après la chirurgie.Dans cette étude, nous avons montré que plusieurs enzymes de la voie sont plus exprimées dans le tissu adipeux des individus obèses que des minces. L’augmentation de l’expression des enzymes dans le tissu adipeux des individus obèses provient d’une part de la présence de macrophages pro-inflammatoires dans le tissu adipeux et également de la réponse des adipocytes aux stimuli pro-inflammatoires. En parallèle, nous avons montré que les concentrations circulantes des kynurénines et le ratio kynurénine sur tryptophane augmentent avec l’IMC et qu’ils diminuent un an après la chirurgie bariatrique. Dans notre étude, comme attendu, la chirurgie bariatrique est associée à une amélioration voire à une rémission du diabète. Nous avons montré également que le maintien des concentrations d’acide kynurénique et d’acide quinolinique sont associés respectivement à la rémission du diabète et à l’amélioration des traits cliniques qui définissent le diabète. La diminution des concentrations en acide xanthurénique après la chirurgie est associée au contraire à une amélioration des traits cliniques qui définissent le diabète. / Tryptophan, an essential amino acid, is either used in protein synthesis or metabolized via the serotonin or the kynurenine pathway. The kynurenine pathway is the main route of tryptophan degradation and generates several metabolites collectively called “kynurenines”. The expression of kynurenine pathway enzymes is induced by inflammatory mediators. Consequently kynurenine synthesis could be induced in individuals with obesity. In fact, obesity is characterized by a chronic low grade inflammation of the adipose tissue reflected by increased serum levels of inflammatory factors which are known to contribute to the development of obesity-induced insulino-resistance. Some metabolites of the kynurenine pathway have been proposed to be risk factors for the development of insulin resistance. Bariatric surgery is currently the most effective treatment for severe obesity and results in a significant weight loss, a decreased level of inflammatory factors and an amelioration of glucose homeostasis. The first enzyme of the kynurenine pathway, IDO1, is known to be more expressed in the adipose tissue of individuals with obesity compared to lean individuals. The kynurenine over tryptophan ratio reflects the activity of IDO1 and is also increased in individuals with obesity.Our objective was to characterize the expression of the kynurenine pathway enzymes in the adipose tissue of women with severe obesity and to evaluate serum levels of the kynurenine pathway metabolites to determine whether these factors could be associated with the appearance of diabetes. This study was performed in women with severe obesity with or without type 2 diabetes. Then we investigated the consequences of weight loss induced by bariatric surgery on levels of circulating kynurenines in order to evaluate whether these variations could explain the improvement in glucose control and type 2 diabetes remission after one year follow-up.In this study, we have shown that several kynurenine pathway enzymes were more expressed in the adipose tissue of women with obesity compared to lean controls. This increase is due to the presence of pro-inflammatory macrophages in the adipose tissue and also comes from the adipocyte response to inflammatory stimuli. In addition, we observed that the serum level of kynurenine and kynurenine over tryptophan ratio are higher in women with higher BMI and they both decrease one year after bariatric surgery. In addition, we observed that the serum level of kynurenine and kynurenine over tryptophan ratio are higher in women with higher BMI and they both decrease one year after bariatric surgery. As expected, bariatric surgery is associated with the improvement and even the remission of type 2 diabetes. We have shown that higher levels of kynurenic acid and quinolinic acid one year after the surgery are associated respectively with type 2 diabetes remission and better glucose homeostasis and that lower levels of xanthurenic acid are associated with better glucose homeostasis.

O melanoma é composto por células malignas e também por um estroma de sustentação que inclui fibroblastos, células imunológicas, endoteliais, matriz extracelular, dentre outros fatores. Assim, os tumores não são entidades independentes, eles interagem ativamente com o microambiente adjacente de forma bidirecional através de sinais moleculares que modulam o fenótipo maligno. Um dos sinais bioquímicos para desenvolvimento desse fenótipo se dá pelo catabolismo de Trp pela via das quinureninas, que gera compostos com diversas atividades biológicas, que no tumor estão envolvidas com tolerância e imunoescape e, logo, com prognóstico ruim para os pacientes. Até o presente momento apenas o consumo de Trp e a formação de um único metabólito, a quinurenina (KYN), tem sido associada a malignidade dos melanomas. A fim de ampliar e elucidar os mecanismos bioquímicos do metabolismo desse aminoácido em melanomas, estudamos mais de quinze compostos de todas as rotas catabólicas de Trp em células da pele, células imunológicas, linhagens tumorais e amostras clínicas de melanoma. De forma inédita pudemos observar que as células da pele tem maior habilidade de sintetizar KYN quando comparadas às linhagens tumorais, demonstrando que o catabolismo de Trp peritumoral pode ser responsável pelos fenômenos de imunotolerância e escape. Além disso, o metabolismo de Trp pode estar envolvido nos mecanismos de homeostasia da pele, já que especificamente essas células produzem compostos com atividade biológica nesse órgão. As células imunológicas possuem um perfil metabólico completamente diferente umas das outras: monócitos, macrófagos e dendríticas possuem maior ativação da via KYN enquanto linfócitos e neutrófilos possuem maior indução da rota que gera serotonina e melatonina. Mesmo nos diferentes fenótipos de macrófagos, M1 e M2a, foram observadas marcações especificas de metabolismo, que podem estar relacionadas às atividades anti- ou pró-tumoral dessas células no microambiente. Em amostras clínicas, apesar da principal diferença entre nevos e melanomas ser a concentração de KYN, diversas outras alterações no metabolismo de tiptofano foram observadas, o que mostra a complexa magnitude deste metabolismo na fisiopatologia da pele / Melanoma is composed of malignant cells and also by a stromal support that includes fibroblasts, immune cells, endothelial cells, extracellular matrix, among other factors. Thus, tumors are not separate entities; they actively interact with the surrounding microenvironment bi-directionally through molecular signals that modulate the malignant phenotype. One of biochemical signals for the development of this phenotype occurs by Trp catabolism through kynurenine pathway, that generates compounds with diverse biological activities, which in tumors are involved with tolerance and imunoescape and therefore with poor prognosis for patients. To date only the consumption of Trp and formation of a single metabolite, kynurenine (KYN), has been associated with malignant melanomas. In order to enlarge and clarify the biochemical mechanisms of this amino acid metabolism in melanomas, we have studied more than fifteen compounds of all catabolic routes of Trp in skin cells, immune cells, tumor cell lines and clinical samples of melanoma. In an unique way we could observe that the skin cells has superior ability to synthesize KYN when compared to tumor cell lines, demonstrating that the peritumoral catabolism of Trp may be responsible for the phenomena of immune tolerance and escape. Furthermore, the Trp metabolism may be involved in skin homeostasis mechanisms, since these cells produce specific compounds with biological activity in this organ. The immune cells have a completely different metabolic profile among them: monocytes, macrophages and dendritic cells have greater KYN pathway activation, and lymphocytes and neutrophils possess greater induction of the route that generates serotonin and melatonin. Even in different macrophages phenotypes, M1 and M2a, we observed specific metabolic marks, which may be related to the anti- or pro-tumoral activity of these cells in the tumor microenvironment. In clinical samples, although the main difference between nevi and melanomas is the concentration of KYN, a range of other changes in Trp metabolism were observed, which shows the complex magnitude of this metabolism in the skin pathophysiology

Nanog is a transcription factor that plays a central part in the gene regulatory network that maintains and induces pluripotency of embryonic stem cells (ESCs). However, the molecular basis by which Nanog achieves its functions is not fully understood. At the centre of C-terminal domain of Nanog a tryptophan repeat (WR) is located, comprising 10 penta-peptide repeats each starting with a tryptophan. A mutant form of Nanog (Nanog-W10A) in which all 10 tryptophan residues have been substituted by alanine has an impaired capacity to drive LIF-independent self-renewal and a reduced efficiency in reprogramming primed epiblast stem cells to naïve pluripotency. To understand how the WR contributes to Nanog function, Nanog-W10A-ERT2 was introduced into Nanog null cells. Upon hydroxytamoxifen addition, the Nanog-ERT2 fusion proteins were detected on chromatin within 1 hour, allowing a comparison of genome-wide transcriptional responses to Nanog and Nanog-W10A by microarray. When treated with LIF, Nanog-W10A can activate most of Nanog targets as efficiently as Nanog. In contrast, Nanog-W10A did not efficiently repress most Nanog targets, including Otx2 and Tcf15 that were previously suggested to prime ESCs for differentiation. The microarray experiments performed in the absence of LIF signalling showed that Nanog and LIF co-regulate an extensive list of targets, including Klf4 and Mras. When LIF is absent, wildtype Nanog can still activate pro-self-renewal factors, including Esrrb and repress differentiation-priming factor, such as Tcf15 and Otx2. In contrast, in the absence of LIF, the activation of pro-self-renewal factors Klf4 and Mras is reduced. In addition, activation of Esrrb by Nanog-W10A induction delays but does not prevent differentiation. These effects allow the de-repression of Otx2 and Tcf15 by Nanog-W10A to dominate. Therefore, the function of Nanog is not only mediated by the activation of pro-self-renewal genes, but also repression of pro-differentiation signals. The functional significance of the repression of Nanog targets was further exemplified by the robust capacity of Otx2 to dominate over the self-renewal signals and to drive differentiation. The Otx2 protein is a direct interacting partner of Nanog that binds the Nanog WR tryptophan residues. The previously identified Otx2 “tail domain” comprises two imperfectly aligned repeats and aromatic residues of each repeat align with aromatic residues of the Sox2 “SXS/TY” motif previously identified to mediate the interaction between Sox2 and Nanog. Aromatic residues of Otx2 were demonstrated to directly interact with both Nanog and Sox2. The interactions between Otx2, Nanog and Sox2 are essential for Otx2 functions in driving ESCs differentiation, as Otx2 mutants with alanine substitutions of the aromatic residues in both or either of the repeats have reduced efficiency to drive differentiation. As Nanog and Sox2 may co-occupy many loci important in maintaining ESC self-renewal, Otx2 may be able to “read” the Nanog/Sox2 co-binding sites to dissolve the pluripotent networks. In summary, the repression function of Nanog is located within the Nanog WR region and represents an important module of Nanog in fine-tuning the balance between self-renewal and differentiation. This module involving Nanog WR can also be recognised by differentiation-priming factor Otx2 and may represent an initial step during the exit of differentiation.

Disruptions in brain serotonin (5-hydroxytryptamine, 5-HT) signaling pathways have been associated with etiology and pathogenesis of various neuropsychiatric disorders, but specific neural mechanisms of 5-HT function are yet to be fully elucidated. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme for brain 5-HT synthesis. Therefore, in this study a tamoxifen (Tam)-inducible cre-mediated conditional gene (Tph2) knockout in adult mouse brain (Tph2icKO) has been established to decipher the specific role of brain 5-HT in the regulation of behavior in adulthood. Immunohistochemistry and high-performance liquid chromatography (HPLC) were used first to test the efficacy of Tam-inducible inactivation of Tph2 and consequential reduction of 5-HT in adult mouse brain. Tam treatment resulted in ≥90% reduction in the number of 5-HT immuno-reactive cells in the anterior raphe nuclei. HPLC revealed a significant reduction in concentration of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) in selected brain regions of Tph2icKO, indicating the effectiveness of the protocol used. Second, standard behavioral tests were used to assess whether reduced brain 5-HT concentrations could alter anxiety-, fear- and depressive-like behavior in mice. No altered anxiety- and depressive-like behaviors were observed in Tph2icKO compared to control mice (Tph2CON) in all indices measured, but Tph2icKO mice exhibited intense and sustained freezing during context-dependent fear memory retrieval. Tph2icKO mice also exhibited locomotor hyperactivity in the aversive environments, such as the open field, and consumed more food and fluid than Tph2CON mice. Lastly, the combined effect of maternal separation (MS) stress and adult brain 5-HT depletion on behavior was assessed in male and female mice. Here, MS stress, 5-HT depletion and their interaction elicited anxiety-like behavior in a sex-dependent manner. MS reduced exploratory behavior in both male and female mice. Reduced 5-HT enhanced anxiety in female, but not in male mice. Furthermore, expression of genes related to the 5-HT system and emotionality (Tph2, Htr1a, Htr2a, Maoa and Avpr1a) was assessed by performing a quantitative real-time PCR. In Tph2icKO mice there was a reduction in expression of Tph2 in the raphe nuclei of both male and female mice. Interaction between MS stress and 5-HT deficiency was detected showing increased Htr2a and Maoa expression in raphe and hippocampus respectively of female mice. In male mice, MS stress and 5-HT depletion interaction effects reduced Avpr1a expression in raphe, while the expression of Htr1a, Htr2a and Maoa was differentially altered by 5-HT depletion and MS in various brain regions. / Unterbrechungen der Serotonin-Stoffwechselwege (5-Hydroxytryptamin, 5-HT) im Gehirn wurden mit der Ätiologie und der Pathogenese von verschiedenen neuropsychiatrischen Erkrankungen assoziiert, wobei die neuronalen Mechanismen der 5-HT Funktionen noch vollständig entschlüsselt werden müssen. Die Tryptophan-Hydroxylase 2 (TPH2) ist das limitierende Enzym für die 5-HT Synthese im Gehirn, weshalb der durch Tamoxifen (Tam) induzierbare, cre-vermittelte Tph2 Gen-Knockout (Tph2icKO) im adulten Mausgehirn möglicherweise helfen könnte die spezifische Rolle von 5-HT im Gehirn in der Regulation von adultem Verhalten zu entschlüsseln. Zuerst wurden Hochleistungsflüssigkeitschromatographie (HPLC) und Immunhistochemische Analysen durchgeführt um die Effizienz der Tam induzierten Inaktivierung des Tph2 und die daraus folgende Reduktion von 5-HT im Gehirn zu überprüfen. Die Behandlung mit Tam resultierte in einer ≥86% Reduktion der Anzahl von 5-HT immunoreaktiven Zellen in der anterioren Raphe im Gehirn. Die HPLC zeigte eine signifikante Reduktion der 5-HT Konzentration und dessen Stoffwechselprodukts 5-Hydroxyindolylessigsäure (5-HIAA) in ausgewählten Gehirn regionen von Tph2icKO, was auf die Effektivität des benutzten Protokolls hindeutet. Danach wurden standarisierte Verhaltens tests durchgeführt um festzustellen, ob eine reduzierte 5-HT Konzentrationen im Gehirn zu einer Veränderung in der Angstreaktion, Depression und im Furchtverhalten der Mäuse führt. Bei allen Tests konnte sowohl in den Tph2icKO-Mäusen als auch in den Kontrolltieren kein offensichtliches angstbezogenes und depressionsähnliches Verhalten festgestellt werden, wobei die Tph2icKO-Mäuse intensive und anhaltende Furcht im Kontext „dependent fear retrieval“ zeigten. Tph2icKO-Mäuse zeigten zudem lokomotorische Hyperaktivität und konsumierten mehr Futter und Flüssigkeit als die Kontrolltiere. Zuletzt wurde der kombinierte Effekt von Stress durch mütterliche Trennung (MS) und adulter 5-HT Reduktion im Gehirn auf das Verhalten von männlichen und weiblichen Mäusen untersucht. Wieder rief nicht der depressionsähnliche Phänotyp, sondernder Stress durch die mütterliche Trennung (MS) und 5-HT Verarmung und deren Interaktion ein angstähnliches Verhalten in Abhängigkeit vom Geschlecht hervor. Reduziertes 5-HT vergrößerte die Angst in weiblichen, aber nicht in männlichen Mäusen. Stress durch mütterliche Trennung (MS) reduzierte das explorative Verhalten sowohl in Männchen als auch in Weibchen. Die Expression von Genen, welche im Bezug zum 5-HT System stehen (Tph2, Htr1a, Htr2a, Maoa und Avpr1a) wurden mit Hilfe von quantitativer Real-Time PCR untersucht. Die Tam Behandlung reduzierte dasTph2 Level in der Raphe bei beiden Geschlechtern signifikant. In weiblichen Mäusen steigertedie Interaktion zwischen Stress durch mütterliche Trennung (MS) und 5-HT Verarmung das Htr2a und Maoa Expressions level in der Raphe und im Hippokampus. In männlichen Mäusen reduzierte die Interaktion von Stress durch mütterliche Trennung (MS) und 5-HT Reduktion die Avpr1a Expression in der Raphe. Die Expression von Htr1a, Htr2a und Maoa wurde in verschiedenen Gehirn regionen unterschiedlich von Tam und Mütterliche Trennung MS verändert. In der Amygdala wurde nur ein MS Effekt auf die Tph2 Expression in den Mäusen sichtbar.

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