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<p>The brain serotonergic and dopaminergic systems broadly influence our internal experience and the ways in which we interact with the outside environment, with crucial regulatory roles in mood, sleep, appetite and the control of voluntary movement. Serotonin and dopamine neurons are themselves influenced by a wide variety of internal and external factors, many of which remain poorly understood. The central aim of this thesis was to better characterize several of these modulatory influences via exploratory investigations involving pharmaceutical agents or environmental modification. Specifically, I examined the modulatory effects of selective serotonin reuptake inhibitors (SSRIs), atypical neuroleptics and environmental enrichment with exercise on the regulation of brain serotonin and dopamine neurons.</p> <p>This thesis documents, for the first time, that (1) inhibition of the serotonin transporter (SERT) by SSRIs induces a rapid and region-selective reduction of tryptophan hydroxylase (TPH)-immunoreactive neurons in serotonergic brainstem nuclei that persists over a prolonged treatment course; that (2) selective blockade of SERT by SSRIs can rapidly induce a reduction of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the substantia nigra (SN) and the ventral tegmental area (VTA) that, again, persists over a lengthy treatment course; that (3) environmental enrichment with exercise can potentiate the effect of SERT inhibition on SN dopaminergic neurons, but not the dorsal raphe nucleus (DRN) serotonergic neurons; that (4) that SSRI fluoxetine triggers a significant upregulation of microglia in the SN; that (5) environmental enrichment with exercise can reduce TPH immunoreactivity in the DRN and TH immunoreactivity in the SN and VTA, even in the absence of any pharmacological intervention, and finally, that (6) the atypical neuroleptic risperidone significantly reduces TPH in the DRN of both young and aged animals and reduces DRN Nissl counts in aged animals. Taken together, the body of work included in this thesis suggests that SSRIs, atypical neuroleptics and environmental enrichment with exercise can have profound effects on brain serotonergic and dopaminergic neurons, possibly accounting for some of the side effects and therapeutic benefits associated with these interventions.</p> / Doctor of Philosophy (PhD)

Disabled-2 (Dab2) is an adapter protein that interacts with cell membranes and it is involved in several biological processes including endocytosis and platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane and helps co-localization with clathrin coats. Dab2, released from platelet alpha granules, inhibits platelet aggregation by binding to the °IIb? integrin receptor on the platelet surface through an Arg-Gly-Asp (RGD) motif located within the PTB domain. Alternatively, Dab2 binds sulfatides on the platelets surface, and this binding partition Dab2 in two pools (sulfatide and integrin receptor-bound states), but the biological consequences of lipid binding remain unclear. Dab2 binds sulfatides through two basic motifs located on its N-terminal region including the PTB domain (N-PTB). We have characterized the binding of Dab2 to micelles, which are widely used to mimic biological membranes. These micellar interactions were studied in the absence and presence of Dab2 lipid ligands, sulfatides and PIP2. By applying multiple biochemical, biophysical, and structural techniques, we found that whereas Dab2 N-PTB binding to PIP2 stabilized the protein but did not contribute to the penetration of the protein into micelles, sulfatides induced conformational changes and facilitated penetration of Dab2 N-PTB into micelles. This is in agreement with previous observation that sulfatides, but not PIP2, protect Dab2 N-PTB from thrombin cleavage. By studying the mechanism by which Dab2 targets membranes, we will have the opportunity to manipulate its function in different lipid-dependent biological processes. / Master of Science

No / Vitiligo is characterized by a mostly progressive loss of the inherited skin color. The cause of the disease is still unknown, despite accumulating in vivo and in vitro evidence of massive oxidative stress via hydrogen peroxide (H2O2) and peroxynitrite (ONOO−) in the skin of affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Since depletion of the essential amino acid l-tryptophan (Trp) severely affects various immune responses, we here looked at Trp metabolism and signaling in these patients. Our in vivo and in vitro data revealed total absence of epidermal Trp hydroxylase activities and the presence of H2O2/ONOO− deactivated indoleamine 2,3-dioxygenase. Aryl hydrocarbon receptor signaling is severely impaired despite the ligand (Trp dimer) being formed, as shown by mass spectrometry. Loss of this signal is supported by the absence of downstream signals (COX-2 and CYP1A1) as well as regulatory T-lymphocytes and by computer modeling. In vivo Fourier transform Raman spectroscopy confirmed the presence of Trp metabolites together with H2O2 supporting deprivation of the epidermal Trp pool by Fenton chemistry. Taken together, our data support a long-expressed role for in loco redox balance and a distinct immune response. These insights could open novel treatment strategies for this disease.—Schallreuter, K. U., Salem, M. A. E. L., Gibbons, N. C. J., Maitland, D. J., Marsch, E., Elwary, S., Healey, A. R. Blunted epidermal l-tryptophan metabolism in vitiligo affects immune response and ROS scavenging by Fenton chemistry, part 2: epidermal H2O2/ONOO−-mediated stress in vitiligo hampers indoleamine 2,3-dioxygenase and aryl hydrocarbon receptor-mediated immune response signaling.

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DHS) catalyzes the first step of the shikimate pathway - a pathway involved in Tyrosine (Tyr), Tryptophan (Trp) and Phenylalanine (Phe) biosynthesis - by condensation of phosphoenolpyruvate and erythrose-4-phosphate to DAHP. Our lab previously demonstrated that Arabidopsis thaliana shikimate pathway flux is regulated by Tyr and Trp. This project suggests that A. thaliana DHS1 overexpressor lines have increased Trp accumulation with Tyr treatment, and that an A. thaliana DHS2 overexpressor line treated with Tyr has unchanged Trp accumulation, indicating that AtDHS2 is Tyr-sensitive. Confocal microscopy of all 3 AtDHS isoforms fused to yellow fluorescent protein demonstrates chloroplast localization. Bimolecular fluorescence complementation indicates that protein-protein interactions occur in the cytoplasm, and not in the chloroplast, for AtDHS1 and AtDHS2 with the metabolic regulator At14-3-3ω. These findings suggest that protein-protein interactions could regulate accumulation of AtDHS2 in the chloroplast, and are perhaps modulated by Tyr.

Amino acid availability plays an important role in modulating the activity of T-cells. One of the pathways employed by T-cells to sense nutrient levels is the “mammalian target of rapamycin” (mTOR) pathway that is inhibited in response to nutrient depletion. Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme along the tryptophan catabolising kynurenine pathway. T-cells are very sensitive to lack of this essential amino acid in their microenvironment and this confers strong immunomodulatory properties to cells expressing active IDO. It therefore has a significant physiological role as a homeostatic mechanism used in mammalian organisms to dampen excessive activation of the immune system but is also used as an immune evasion mechanism by many cancers. In this study, we investigated the IDO inhibitory properties and mechanism of action of the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) that potentially forms a negative feedback loop in the kynurenine pathway. We studied the molecule in enzymatic assays, in live cells and discovered that it inhibits IDO in an indirect way via the formation of hydrogen peroxide. Secondly, we looked at the effects of tryptophan and its metabolites on T-cell proliferation and mTOR activity, and discovered a metabolite that inhibits T-cell proliferation. Lastly we examined mechanisms of T-cell suppression employed by myeloid derived suppressor cells (MDSCs), focusing on their ability to deplete amino acids from their microenvironment. We were able to exclude tryptophan consumption as a suppressive mechanism and established that by manipulating extracellular concentrations of several amino acids other than arginine and cysteine – that are known to be utilised by MDSCs - we were able to reduce their inhibitory properties. In summary, we have described in detail how 3-HAA inhibits IDO in in vitro assays, outlined how some tryptophan metabolites can inhibit T-cell proliferation, and clarified aspects of suppressive mechanism employed by MDSCs.

Štěpána Boukalová Mechanisms of activation and modulation of vanilloid TRP channels TRPV1 and TRPV3 are thermosensitive ion channels from the vanilloid subfamily of TRP receptors. TRPV1, which is primarily expressed in nociceptive sensory neurons, is an important transducer of painful stimuli and is also involved in the detection of noxious heat. TRPV3 is expressed mainly in the skin where it regulates proliferation and differentiation of keratinocytes. Similarly to voltage-dependent potassium (Kv) channels, TRP receptors are comprised of four subunits, each with six transmembrane segments (S1-S6). Using mutational approach, we tried to elucidate the role of S1 in TRPV1 functioning. Our results indicate that the extracellular portion of S1 plays a crucial role in TRPV1 gating. TRPV1 channels with a conservative mutation of positively charged residue in this region (R455K substitution) were overactive. However, they were neither activated nor potentiated by low pH; on the contrary, protons stabilized the closed conformation of this mutant channel. Very similar phenotypic properties were found in other TRPV1 mutants with substitution in S4/S5-S5 region and in the pore helix. In Kv channels, extracelular portion of S1 forms a small contact surface with the pore helix, which allows efficient transmission of...

Le tube digestif héberge une communauté microbienne complexe, le microbiote intestinal, dont les capacités métaboliques sont plus riches et diversifiées que celles codées par le génome de l'hôte. L'implication du microbiote intestinal dans divers aspects de la physiologie de l'hôte, comme le métabolisme nutritionnel et l'immunité, est depuis longtemps étudiée. En revanche, l'action potentielle du microbiote sur le développement et le fonctionnement du cerveau constitue une nouvelle piste de recherche, encore peu explorée. Dans ce contexte, nous avons réalisé une première étude générale de l'action du microbiote intestinal sur le cerveau en comparant les fonctions sensori-motrices, le comportement de type anxieux, l'état d'activation de l'axe hypothalamo-hypophyso-surrénalien et le profil cérébral des monoamines de rats F344 axéniques et conventionnels. Les résultats révèlent que, chez cette lignée particulièrement sensible au stress, l'absence de microbiote intestinal exacerbe le comportement de type anxieux et la réponse hormonale au stress, et atténue le métabolisme dopaminergique cérébral. Afin d'étudier par quel moyen le microbiote peut agir sur le cerveau, une seconde étude a été menée, ciblant un métabolite bactérien spécifique, l’indole, dont certains dérivés oxydés par le foie sont connus pour avoir des propriétés neuroactives. L'indole est un métabolite naturel du microbiote intestinal, dont la surproduction pourrait survenir lors d'une dysbiose du microbiote. Deux cas de surproduction ont été modélisés : chronique et aiguë. Dans les deux cas, des modifications importantes du comportement de l'hôte ont été observées. En situation de surproduction chronique, l'indole favorise des comportements de type anxieux et dépressif, tandis qu'une surproduction aiguë a un effet sédatif marqué. D'un point de vue mécanistique, nous confirmons que l’indole peut agir sur le système nerveux central par la voie sanguine impliquant les dérivés oxydés et montrons pour la première fois qu'il peut aussi agir en activant les noyaux cérébraux du nerf vague. / The gastro-intestinal tract hosts a complex microbial community, the gut microbiota, whose collective genome coding capacity vastly exceeds that of the host genome. The involvement of the gut microbiota in various aspects of the host physiology, such as the nutritional metabolism and the immunity, has long been studied. In contrast, the possible action of the gut microbiota on brain development and functioning is a new line of research, still poorly explored. In this context, we performed a first general study of the effect of gut microbiota on the brain by comparing the sensory-motor functions, the anxiety-like behaviour, the activation of the hypothalamic-pituitary-adrenal axis and the brain monoamine profile in germ-free and conventional F344 rats. The results show that, in this particularly stress-sensitive strain, absence of gut microbiota exacerbates the anxiety-like behaviour and neuroendocrine response to stress, and reduces brain dopamine metabolism. To investigate the means by which the microbiota can affect the brain, a second study was conducted, targeting a specific bacterial metabolite, indole, whose oxidative derivatives, produced by the liver, are known to have neuroactive properties. Indole is a natural metabolite of the gut microbiota, whoseoverproduction could occur during a microbiota dysbiosis. Two conditions of overproduction, namely chronic and acute, were modelled. In both cases, significant changes in the behaviour of the host were observed. In chronic overproduction, indole promotes anxiety- and depressive-like behaviours, while acute overproduction has a marked sedative effect. From a mechanistic point of view, we confirm that indole can act on the central nervous system through its oxidized derivatives and show for the first time that it can also act by activating the brain nuclei of the vagus nerve.

Espécies carbonílicas tripletes formadas quimicamente no escuro, por exemplo, durante a peroxidação de lipídios, têm reatividade química análoga à de radicais alcoxilas. Aventou-se que tais espécies possam estar implicadas na fisiopatologia de doenças degenerativas (\"estresse carbonílico\"). A pesquisa dos efeitos de espécies tripletes sobre algumas biomoléculas e consequentes respostas biológicas, propostas e pesquisadas no período 1970 - 1990 (hipótese de \"fotoquímica sem luz\" dos Profs. G. Cilento, IQUSP, e Emil H. White, Johns Hopkins University), encontrou empecilhos instrumentais e relativamente poucas propostas foram confirmadas. Com o uso de técnicas de alta resolução, tais como EPR, HPLC e MS, este trabalho teve como objetivo analisar intermediários e produtos de tais processos e estudar mecanismos de reação de acetona triplete, produzida quimicamente pela decomposição térmica de 3,3,4,4-tetrametildioxetano (TMD) ou, enzimaticamente, pela oxidação aeróbica de isobutanal (IBAL), catalisada por peroxidase de raiz forte (HRP), na presença de aminoácidos e proteínas. Este trabalho demonstra a formação de um radical acetila, presumidamente formado da clivagem &#945; de acetona triplete, e um radical terciário centrado em carbono, formado pela abstração de hidrogênio do IBAL. Resultados de espectrometria de massas demonstraram a formação de três diferentes adutos entre o radical terciário de IBAL, com L-Trp. Aventou-se que um dos produtos era resultante de alteração no nitrogênio e os outros no carbono 3, ambos no anel indólico. Observou-se também a formação de produto correspondente ao radical hidroxipropionil com L-Trp. Também se observaram dois produtos de L-Trp típicos de sua oxidação por oxigênio singlete, a formilquinurenina, e um aduto de função álcool. A formação de base de Schiff entre o L-Trp estudado e o IBAL também é apresentada. A formação de oxigênio singlete foi evidenciada indiretamente via EPR utilizando o spin trap TEMP e através de um captador de adição-9,10 (tipo Diels-Alder) em derivado de antraceno. Foram realizados, também, experimentos com precursores de melanina e demonstrou-se a formação de espécies excitadas do ácido 5,6-dihidroxi-indol-2-carboxílico (DHICA) que poderiam explicar a formação de produtos de DNA tipicamente resultantes de reação fotoquímica, mas na ausência de luz. Tais resultados corroboram a reação de espécies tripletes com biomoléculas, possibilitando a compreensão de número significativo de eventos biológicos conhecidos, mas teoricamente \"proibidos\" de ocorrer no estado fundamental, em tecidos não expostos à luz / Electronically excited triplet carbonyl species formed as products of some biochemical reactions, such as lipid peroxidation, behave similarly as alcoxyl radicals. It has long been hypothesized that such excited species could have a role in some diseases (\"carbonyl stress\"). Research of chemical lesions of triplet carbonyls over biomolecules and their biological response took place principally from 1970 to 1990 (the \"photochemistry without light\" hypothesis proposed by Profs. G. Cilento, IQUSP, and Emil H. White, Johns Hopkins University), but it suffered from the lack of required instrumentation, and just few cases of photo(bio)chemistry without light were confirmed. The aim of this work, using high resolution techniques (EPR, HPLC, and MS), is to analyze the reaction products of excited triplet acetone with aminoacid and protein targets. Triplet acetone was produced from the thermal decomposition of 3,3,4,4-tetramethyldioxetane (TMD) or from the aerobic oxidation of isobutanal (IBAL) catalyzed by horseradish peroxidase (HRP). We revealed the generation of acetyl radical, putatively originated from &#945;-cleavage of triplet acetone, and a carbon-centered tertiary radical, proposed as an IBAL radical formed by hydrogen abstraction from IBAL. Mass spectrometry showed production of three adducts from the reaction of IBAL radical with L-Trp, one of them at the nitrogen 1 and the other two at carbon 3 from the amino acid indole ring. Two adducts with m/z correspondent to the reaction between L-Trp (at carbon 3) and a hydroxypropionyl radical, and two products typically formed from singlet oxygen (formylkynurenine and an alcohol L-Trp adduct) were also observed. A Schiff base between L-Trp and IBAL was also observed. Singlet oxygen production from triplet-triplet energy transfer from excited acetone to ground state molecular oxygen was indirectly showed by EPR spin trapping with TEMP, and by MS using the anthracene derivative EAS to trap (9,10-cycloaddition) of 18O2 (1&#916;g). Other data reported here include the demonstration of excited species formed when DHICA, a melanin precursor, was oxidized. These results might explain the generation of DNA photochemical products (thymine dimers) in the absence of light. Altogether, we collect strong and significant evidence in this thesis that corroborate the reactivity of triplet excited species with a couple of biomolecules, providing insights over some reportedly known molecular events that are theoretically forbidden to occur in the ground state but happen in tissues non-exposed to light

A artrite reumatoide (AR) é a artropatia inflamatória mais prevalente no mundo, de etiologia multifatorial e fenótipos heterogêneos. Busca-se, além de definir fatores etiológicos, compreender as interações entre mecanismos envolvidos na fisiopatologia da AR. Entre estes, fatores genéticos, tanto genes do antígeno leucocitário humano (HLA), especialmente a presença do epítopo compartilhado (Shared epitope - SE) do HLA-DRB1, como genes não-HLA, e fatores ambientais e epigenéticos têm sido associados à doença. Assim, a identificação de novos fatores relacionados à etiopatogenia da AR e suas possíveis associações com características clínicas motivaram esse estudo. Um estudo caso-controle foi desenhado e dividido em duas etapas. Para a primeira etapa, foi obtido plasma de 18 indivíduos de AR e 18 voluntários saudáveis de Ribeirão Preto, no qual foram identificados quinurenina (Kyn), Trp, serotonina (5-HT) e taxa Kyn/Trp (KTR) por cromatografia líquida de ultra-eficiência (CLUE) acoplada a espectrômetro de massas sequencial (CLUE-DAD-EM/EM). Na segunda etapa, de estudo genético, uma coorte formada por 328 indivíduos com AR e por 234 voluntários saudáveis de Ribeirão Preto e de Porto Alegre foi avaliada quanto ao polimorfismo do gene da enzima indoleamine 2,3-dioxigenase 1 (IDO1). Foram obtidos dados clínicos e epidemiológicos e coletadas amostras de sangue periférico para extração de DNA pelo método de salting-out. Em seguida, tipificação HLA e reação em cadeia de polimerase (RCP) das variantes da IDO1, rs7820268, rs3739319, rs61753677, rs35059413, rs35099072 e rs9298586, foram realizadas. A positividade para fator reumatoide (FR) em indivíduos com AR foi associada ao tabagismo (p= 0.0002) e ao SE (p < 0.0001), e para anticorpo antipeptídeo citrulinado cíclico (anti-CCP), associado ao SE (p < 0.0001). Quando combinadas a presença de SE e a de tabagismo, houve associação estatisticamente significante para FR (p < 0.001) e para anti-CCP (p = 0.03). Foram observadas menores concentrações plasmáticas de 5-HT em indivíduos com AR quando comparados a voluntários saudáveis (p =0.006), mas sem diferença para níveis de Trp, Kyn e KTR. Para estes, diferenças apareceram quando avaliados subgrupos. Em indivíduos com AR sem tratamento com drogas modificadoras do curso da doença (DMCDs), os valores plasmáticos de Trp foram menores quando comparados aos em terapia (p = 0.0016), enquanto em pacientes com AR tabagistas os valores de Kyn e KTR foram menores que em pacientes não tabagistas (p = 0.039 e p = 0.032, respectivamente). Não foram identificadas associações estatisticamente significantes entre as variantes genéticas estudadas e o risco de desenvolver AR, nem entre os polimorfismos da IDO1 estudados e a concentração plasmática de Trp, Kyn e 5-HT e KTR. Este estudo não identificou relação das variantes do gene da IDO1 com suscetibilidade para AR. Assim, novos estudos são necessários para que possam ser explicadas as associações encontradas na via das Kyns e na 5-HT em etiopatogenia da AR. / Rheumatoid arthritis (RA) is the most prevalent inflammatory arthropathy in the world, with multifactorial etiology and heterogeneous phenotypes. Besides defining etiological factors, it is sought to understand the interactions between mechanisms in RA pathophysiology. About these, genetic factors, both human leucocity antigen (HLA) genes, especially the HLA-DRB1 Shared epitope (SE) presence, and not-HLA genes, and environmental and epigenetics factors have been associated with the disease. Therefore, the aim of this study was to identify new possible associations between RA clinical features and its etiopathogenesis. A case-control study was designed and it was divides in two phases. The first phase, it was obtained plasma of 18 RA patients and 18 healthy controls from Ribeirão Preto to identify the kynurenine (Kyn), Trp and serotonin (5-HT) concentrations and Kyn/Trp ratio (KTR) by ultra-high performance liquid chromatography coupled to sequential mass spectrometer. The second phase was a genetic study that evaluated a cohort of 328 RA patients and 234 healthy volunteers from Ribeirão Preto and Porto Alegre about the indoleamine 2,3-dioxygenase 1 (IDO1) gene polymorphism. Clinical and epidemiological data were obtained and peripheral blood samples were collected to DNA extraction by salting-out method. Then, HLA typification and polymerase chain reaction to identify IDO1 genetic variants rs7820268, rs3739319, rs61753677, rs35059413, rs35099072 and rs9298586 were performed. Rheumatoid factor (RF) positivity was associated to smoking (p = 0.0002) and SE (p < 0.0001), and cyclic citrullinated peptide autoantibodies (anti-CCP) positivity was associated to SE (p < 0.0001). When SE presence and smoking were combined, there was statistically significant association to RF (p < 0.001) and anti-CCP (p = 0.03). We observed lower plasma 5-HT concentrations in RA patients than in healthy volunteers (p = 0.006), but no significant difference to Trp, Kyn and KTR levels. For these, differences were observed when subgroups were evaluated. In RA patients not using disease modifying antirheumatic drugs (DMARDs) the plasma Trp levels were lower than RApatients using DMARDs, while the plasma Kyn concentrations and KTR in smokers RA patients were lower than nonsmokers RA patients (p = 0.039 and p = 0.032 respectively). We did not indetify statistically significant associations neither between studied genetic variants and risk to develop RA nor between IDO1 polymorphisms and plasma Trp, Kyn, 5HT concentrations and KTR. This study did not identify relation between IDO1 genetic variants with susceptibility to RA. Therefore, new studies are necessary to explain the searched associations between Kyns pathway and 5-HT in RA etiopathogenenesis.