<p>  </p> <p>In commercial settings, piglet weaning and transportation occur concurrently due to the expansive application of multi-site production systems across the United States and the combination of these events can be defined as an early life stressor. Early life stress is known to reduce pig performance, efficiency, and immune resilience contributing to reduced welfare and increased production losses. To combat the deleterious effects of stress on pigs, the essential amino acid tryptophan (Trp) supplemented above current National Research Council (NRC 2012) recommended levels, improves neuroendocrine responses to stress, lowers plasma cortisol and norepinephrine concentrations, and improves hypothalamic-pituitary-adrenal axis recovery time following stress. However, there are discrepancies concerning the Trp requirement for nursery pigs, suggesting the 2012 recommendations for Trp may be inaccurate for optimizing growth performance and health in modern pigs. Three experiments were conducted to determine the effects of supplemented standard ileal digestible Trp above NRC (2012) recommended levels on performance, feed efficiency, immune vigor, and stress tolerance. The objective of experiment 1 was to eliminate or reduce short- and long-term, transport-induced reductions in piglet feed efficiency and growth by supplementing Trp above NRC (2012) recommendations pre-weaning and/or during the nursery phase. An oral gavage of Trp or a control milk carrier was provided to pre-allotted piglets beginning at day 5 of lactation and continuing to weaning. At weaning all pigs were blocked by sex, weaning weight and pre-wean treatment and randomly assigned to transport and post-wean treatments. Pigs were fed in four nursery phases with diets containing 1X or 2X NRC recommended concentrations of SID Trp and a common grower diet fed in 6 phases during the grow-finish period. At market, loin characteristics were measured via ultrasound and carcass data was collected from the packing plant. Pre-weaning Trp supplementation had no effect on pre-weaning growth performance; however, post-wean Trp increased overall body weight and average daily gain in nursery pigs when Trp was supplemented pre-wean. The objective for experiment 2 was to mitigate the adverse effects of early life transport stress on subsequent immune challenges by providing supplemental Trp during the nursery period. At weaning, pigs were transported for 8 hours and assigned to treatments of vaccine-induced immune challenge and dietary treatment. Pigs were fed standard nursery diets, in four phases, over 35 days with pigs receiving 1X or 2X the NRC (2012) recommended Trp concentration. Half the pigs on each dietary treatment were subjected to a 3-wk vaccine challenge consisting of circovirus, mycoplasma, and influenza vaccines administered in wk 2, 3, and 4 post-wean, respectively. At market, loin characteristics were measured via ultrasound and carcass data was collected.  At the culmination of the nursery period, unchallenged pigs supplemented with Trp were heavier compared to control pigs. This resulted from an overall improvement in average daily gain for 2X Trp fed pigs. However, increasing Trp did not affect market weight, loin eye area, or lean percent in market pigs, although 2X Trp increased back fat. The objective of experiment 3 was to determine what ratio of SID tryptophan, relative to lysine, maximizes growth performance and feed efficiency in weaned pigs during the nursery period. Pigs were blocked by sex and weaning weight and randomly allotted to one of five dietary treatments with Trp increasing stepwise by 0.33%-units, respectively. Pigs were fed standard nursery diets, in four phases, over 35 days with pigs receiving 1X, 1.33X, 1.66X, 2X, or 2.33X the NRC (2012) recommended Trp concentration.  Overall, there were no differences in performance or efficiency across dietary treatments during a 35-day nursery period in unstressed healthy pigs.  In conclusion, increasing Trp in swine diets prior to and/or following stress events can mitigate stress-related perturbations in performance and efficiency.      </p>

Bluthochdruck ist ein globales Gesundheitsproblem, das Milliarden von Menschen weltweit betrifft und schwerwiegende physische, finanzielle und auch produktivitätsbezogene Auswirkungen hat (Forum and Health, 2011; WHO, 2013; Williams et al., 2018). Die Aufnahme bioaktiver Peptide mit blutdrucksenkender Wirkung wie das Isoleucin-Tryptophan (IW) ist als mögliche Präventionsstrategie für die Hypertonie in Diskussion. Das Dipetid IW hemmt das Angiotensin-Converting-Enzym (ACE) und zeigt im Tierversuch eine blutdrucksenkende Wirkung (Martin et al., 2008b; Kaiser et al., 2016). Die Absorption dieses Dipeptids ist jedoch noch nicht aufgeklärt. Daher sollte in der vorliegenden Arbeit die Absorption und Stabilität bioaktiver Peptide am Beispiel des Dipeptids IW untersucht werden. Die folgenden 3 Zielsetzungen sollten dabei helfen die Aufnahme von IW in vivo aufzuklären. 1) Untersuchung der intestinalen Absorption von IW ex-vivo mittels Darmperfusion (EIPM). 2) Bewertung der intestinalen Stabilität von IW mittels Darm/Peptid-Co-Inkubation und anschließenden Studien zur Identifikation IW-abbauender Enzyme. 3) Untersuchung auf mögliche weiter gastrointestinale Absorptionsrouten von IW beim Menschen. Im ersten Schritt wurde die ex-vivo Darmperfusions-Methode (EIPM) etabliert, so dass eine physiologische Abschätzung der Absorption von IW durch den Mausdarm möglich wird. Mit dieser Methode wurden die absorptiven Funktionen des Darms ex-vivo zunächst mit den Kontrollsubstanzen, Koffein und Erythrozyten geprüft, bevor die Absorption der Dipeptide IW und WL untersucht wurden. Die Ergebnisse aus den Dipeptid Studien zeigten, dass IW und WL nicht intakt aus dem Mausdarm absorbiert wurden, allerdings die in der Sequenz der Dipeptide enthaltene Aminosäure Tryptophan konnte nachgewiesen werden. Dies lässt darauf schließen, dass die Dipeptide durch die Darmenzyme mit anschließender Freisetzung von Tryptophan rasch abgebaut wurden. Die Berechnung der Massenbilanz zeigten für IW und WL ein signifikantes Massendefizit (18 % für IW und 15 % für WL). Die Darm/Peptid-Co-Inkubationsstudien zeigten ebenfalls eine rasche Abnahme (innerhalb von 5 Minuten) der IW- und WL-Konzentrationen und gleichzeitig eine Zunahme der Tryptophankonzentration. Damit konnte bestätigt werden, dass IW und WL im Darm rasch abgebaut wird. Auch in den Co-Inkubationsstudien gab es wie bei den EIPM-Ergebnissen ein signifikantes Massendefizit (19 % für IW und 21 % für WL). Für die Identifizierung der IW abbauenden Enyzme wurden die Darm-Peptid-Co-Inkubation zusätzlich neben IW mit verschiedenen allgemeinen und spezifischen Inhibitoren durchgeführt. Dabei konnte gezeigt werden, dass Membrandipeptidase, Aminopeptidase W und Aminopeptidase N die Enzyme sind, die hauptsächlich IW im Darm abbauen. Die gleichzeitige Hemmung dieser Enzyme verhinderte signifikant die Hydrolyse von IW im Darm. So wurden 64,1 % des eingesetzten IW nach 5 min quantifiziert. Für Aminopeptidase N und die Membrandipeptidase sind kommerziell erhältliche Antikörper vorhanden, so dass die beiden mittels Western Blot und Immunchemie als Bürstenrandenzyme im Darmepithel nachgewiesen wurden. In einer weiteren Versuchsreihe wurde untersucht, ob die oben genannten Enzyme auch für den Abbau von IW im Plasma verantwortlich sind. Die Ergebnisse zeigten, dass diese identifizierten Enzyme den Abbau von IW im Plasma nicht verhinderten, was zeigt, dass dort andere Enzymgruppen beteiligt sind. Weiter wurden Veränderungen der intestinalen ACE-Aktivität und des intestinalen ACE-Proteinspiegels nach der Inkubation mit IW untersucht. Es zeigten sich durch die Inkubation mit IW eine Verringerung der ACE-Aktivität, die allerdings auf Grund der hohen Standardabweichung keine Signifikanz erreichte. Der ACE-Proteinspiegel zeigte durch die Inkubation mit IW keine signifikanten Veränderungen. Im Hinblick auf die Humanstudien wurde die IW Stabilität in Speichelproben von 4 Freiwilligen untersucht. Während bei einer Probe keinerlei Abbau von IW über 10 min gesehen wurde, zeigten die anderen 3 dagegen einen deutlichen Abbau von IW. Nach 10 min Inkubation waren im Mittel noch 81,7 % IW intakt nach 30 min nur noch 68,6 %. Parallel zum IW – Abbau nahm die Tryptophan-Konzentration zu. Mit der erhaltenen Massenbilanz von 108,2 % konnte eindeutig ein Abbau von IW bestätig werden. Um die mögliche Absorption von IW über den gesamten Magen-Darm-Trakts beim Menschen zu beurteilen, wurde IW über verschiedene Wege verabreicht: sublingual, in Trinkwasser gelöst, als magensaftresistente Kapseln und als nicht magensaftresistente Kapseln. Die Ergebnisse der Absorptionsstudien mit 5 bis 6 Probanden zeigten einen Anstieg von IW im Plasma im Vergleich zu den basalen Werten bei den folgenden Interventionen: als magensaftresistente Kapseln (Mittelwert; +/-304 %, Median; +/-156 %), sublingual (Mittelwert; +/-303 %, Median; +/-204 %), in Trinkwasser gelöst (Mittelwert; +/-266 %, Median; +/-287 %) und schließlich als Kapseln ohne Magensaftresistenz (Mittelwert; +/-163 %, Median; +/-152 %). Alle Messparameter unterlagen hohen interindividuellen Schwankungen. Die Fläche unter den Kurven für den IW-Anstieg wurde in folgender Reihenfolge kalkuliert: IW magensaftresistente Kapseln > sublinguale IW > IW in Trinkwasser gelöst > IW Kapseln ohne Magensaftresistenz. Dies gilt sowohl für den Vergleich des Mittelwerts als auch für den des Median. Eine entsprechende Hemmung der Plasma-ACE-Aktivität wurde auch bei den Teilnehmern nach der IW-Einnahme beobachtet. IW, das über dem sublingualen Weg verabreicht wurde, bewirkte die stärkste Hemmung der Plasma-ACE-Aktivität (Mittelwert; +/-19,1 %, Median; +/-17. 9 %), gefolgt von in Trinkwasser gelösten IW (Mittelwert;+/-16,8 %, Median; +/-18,6 %), dann IW-magensaftresistente Kapseln (Mittelwert; +/-13,6 %, Median; +/-15,1 %) und schließlich IW- Kapseln ohne Magensaftresistenz (Mittelwert; -9,3 %, Median; -9,2 %). Zusammenfassend haben die Ergebnisse der vorliegenden Studie gezeigt, dass IW im Darm rasch abgebaut wird, was für seine Bioverfügbarkeit eine Rolle spielt. Als die wichtigsten Enzyme, die IW im Darm abbauen, wurden die Membrandipeptidase, Aminopeptidase N und Aminopeptidase W identifiziert, diese sind allerdings nicht für den IW-Abbau im Plasma verantwortlich. Schließlich zeigten die Ergebnisse der Humanstudien, dass IW vor allem sublingual und intestinal, in geringerem Masse auch über den Magen absorbiert wurde. Parallel zu der Absorption wurde das Plasma-ACE gehemmt. Diese Studien liefern damit wichtige Grundlagen für die Absorption bioaktiver Peptide, welche für die Überlegungen der Applikationsart dieser Peptide hilfreich sind.

Islet transplantation is a possible curative treatment for type 1 diabetes (T1D). Currently the liver dominates as implantation site, despite the many challenges encountered at this site. Acute hypoxia in islets transplanted to muscle and omentum, two possible alternative sites, was prevailing. However, it was rapidly reversed at both implantation sites, in contrast to when islets were transplanted intraportally. At the intramuscular site hypoxia was further relieved by co-transplantation of an oxygen carrier, polymerized hemoglobin, which also improved the functional outcome. The complement system was activated after islet transplantation to muscle, but did not hamper graft function. Both mouse and human islets transplanted to omentum become well re-vascularized and have a functional blood flow and oxygenation comparable with that of endogenous islets. Animals transplanted with islets to the omentum had a superior graft function compared with animals receiving intraportal islet grafts. Alloxan-diabetic animals were cured with a low number of islets both when the islets were implanted in the omentum and muscle. The islet grafts responded adequately to both glucose and insulin and displayed a favorable mRNA gene expression profile. A challenge in diabetes research and in islet transplantation is that there are no established techniques for quantifying beta-cell mass in vivo. By using radiolabeled Exendin-4, a GLP-1 receptor agonist, beta-cell mass after transplantation to muscle of mice was quantified. The results may well be translated to the clinical setting. By comparing the pancreatic accumulation of [11C]5-hydroxy tryptophan ([11C]5-HTP) as detected by positron emission tomography (PET) in T1D patients with that of healthy controls, a 66% decrease was observed. This may in fact represent the loss of beta-cells, taking into account that other cells within the islets of Langerhans are largely unaffected in T1D.  In conclusion, the data presented support the use of alternative implantation sites for islet transplantation. In addition to improving the functional outcome this may enable more transplantations since the number of transplanted islets may be reduced. The techniques investigated for quantifying transplanted and endogenous beta-cell mass may greatly improve our knowledge of the pathophysiology of T1D and become a valuable tool for evaluation of beta-cell mass.

The growing prevalence of antibiotic-resistant bacteria is a global health crisis that threatens the effectiveness of antibiotics in medical treatment. Increases in the number of antibiotic-resistant bacteria and a drop in the pharmaceutical development of novel antibiotics have combined to form a situation that is rapidly increasing the likelihood of a post-antibiotic era. The development of antibiotics with novel enzymatic targets is critical to stall this growing crisis. In silico methods of molecular modeling and drug design were utilized in the development of novel tryptamine analogs as potential antibiotics through their inhibition of the bacterial enzyme tryptophan synthase. Following the creation of novel tryptamine analogs, the molecules were analyzed in silico to determine their binding affinity to human MAOB and the E. coli α-subunit, E. coli β2-dimer and the M. tuberculosis β2-dimer of tryptophan synthase. Ten tryptamine analogs displayed significant increases in tryptophan synthase binding affinity and show promise as potential antibiotics and antibiotic adjuvants. Further in silico modeling determined that the binding sites of the tryptamine analogs were similar to wild-type tryptamine in the E. coli β2-dimer, the M. tuberculosis β2-dimer and human MAOB, while the analogs’ binding sites to the E. coli α-subunit differed. Although no tryptamine analogs increased tryptophan synthase binding affinity while decreasing human MAOB binding affinity, related increases in MAOB binding affinity warrants further research into the analogs’ potentials as MAO inhibitors. Given the increases in tryptophan synthase binding affinity and similar β2-dimer binding sites, a provisional patent was filed on the ten identified tryptamine analogs. Moving forward, we recommend the synthesis of the ten identified tryptamine analogs. Following synthesis, further research should be conducted to determine the in vitro and in vivo antibiotic properties of the ten tryptamine analogs.

Ternary systems comprising DNA/RNA, proteins and one (or more) metal ion are generating increased interest due to its biological relevance. The knowledge gained from the study of these systems could provide important clues regarding the precise mechanism for transcription factors, repair proteins and metal complexes with anti-tumoral/anti-viral activities.The interactions occurring among the components of these ternary systems can be broadly grouped into covalent and non-covalent. The first kind of interactions can lead to the irreversible transformation of the components in the system, while the second is thought to be reversible leading to transient states and fluxionality. Both kinds of interaction are generally present in living systems, complementing the function of each other.Monofunetional Platinum-nucleobase complexes (MPNs) are synthesized via substitution of a chloride ligand by a nucleobase in platinum complexes with trans geometry. MPNs are particularly interesting for the study of ternary systems since they mimic the first step in the formation of a platinum-DNA adduct and their interaction with aminoacids/proteins provide a good first approach for more complex systems.The presence of the nucleobase as a ligand, significantly modifies the biological activity of these complexes by reducing its cytotoxicity and generating a promising anti-viral activity, especially against HIV-1 virus. The specific role of the nucleobase ligand on these complexes as a non-covalent motif, important for protein recognition, was explored in models involving tryptophan/N-acetyl tryptophan and a small protein domain called zinc finger, containing also a tryptophan residue.The coordination of the nucleobase to a metal ion such as Pt(II) or Pd(II) was found to increase its π-stacking interaction towards aromatic residues in proteins, specifically tryptophan. The enhancing effect was found to depend on the nature of the metal ion, nature of nucleobase and size/complexity of the protein model. Furthermore, DFT studies revealed an important change in the energy for the lowest unoccupied molecular orbital (LUMO) in the coordinated nucleobases, which could place this orbital in an favored position to interact with the highest occupied molecular orbital (HOMO) in the tryptophan residue. Results from calculations showed a good correlation with experimental evidence and could indicate an important role for the frontier molecular orbitals (HOMO/LUMO) of the species involved in the π-stacking interaction.This study was extended to a zinc finger domain from an essential protein in HIV-1 virus, i.e. nucleocapsid protein NCp7. Findings showed that the nucleobase ligand in addition to modulate hydrolysis and reaction rates for MPNs can also be responsible for an initial non-covalent recognition towards a specific protein. This initial recognition has been proposed as the first stage in a two-step mechanism of action for these platinum complexes that ultimately can lead to zinc ejection from the zinc finger domain in the viral NCp7. The significance of the data presented show that is possible to modulate the ligand coordination sphere in metal complexes to can result in great differences in terms of biological effects.The novel chemistry derived from DNA adducts with platinum complexes with a trans geometry was also explored in silico. The molecular dynamics of two free DNA 20-mer is compared with the corresponding metallated-adducts, namely monofunctional, 1,2-bifunctional interstrand and 1,3-bifunctional intrastrand. The differences in terms of structure and energy are compared for these systems, in general the monofunctional adduct exhibited the most interesting feature in terms of structural change in the DNA double strand causing the destacking of the metallated nucleobase. Bifunctional adducts exhibited loss of Watson-crick bonds and localized change in sugar puckering. These results showed that important differences can be found for platinated DNA even at short simulation times < 1 ns.

Depuis l'avènement des techniques d'ionisation douces comme le MALDI ou l'ESI, la spectrométrie de masse est devenue un outil incontournable pour l'étude des biomolécules, et en particulier, des protéines. Le mécanisme d'ionisation spécifique des sources MALDI introduit un phénomène de discrimination spectrale généralement considéré comme un frein. Le greffage d'un motif α-cyano 4-hydroxy cinnamique ou HCCA sur un peptide permet d'en tirer parti en créant une discrimination spectrale favorable au composé marqué. Deux applications du marquage HCCA ont été développées au cours de ce travail de thèse.Une des limitations des études phosphoprotéomiques réside dans la faible ionisation des peptides phosphorylés. De nombreuses méthodes de purification ont été proposées pour contourner ce problème. Nous avons étudié une approche alternative qui consiste à greffer spécifiquement un motif HCCA sur une position phosphorylée afin d'amplifier les signaux des peptides d'intérêt. Nous avons développé en parallèle une méthodologie pour l'étude de la liaison d'un ligand peptidique à son récepteur ne nécessitant pas de radioactivité. Le greffage covalent d'HCCA sur un ligand nous a permis de le détecter et de le quantifier dans une expérience de déplacement. Cette méthodologie a notamment permis de déterminer l'affinité d'un ligand de référence pour le récepteur V1A à la vasopressine. / Since the advent of soft ionization techniques such as MALDI or ESI, mass spectrometry has become an indispensable tool for the study of biomolecules, and particularly proteins. The specific ionization mechanism of MALDI leads to a spectral discrimination phenomenon generally regarded as a restriction. The α-cyano 4-hydroxy cinnamic acid (HCCA) pattern grafting on a peptide can take advantage by introducing a spectral discrimination in favor of the labeled compound. Two applications have been developed during this thesis, based on HCCA tagging.A limitation of phosphoproteomic studies is the low ionization of phosphorylated peptides. Many purification methods have been reported to circumvent this problem. We studied an alternative approach which consists in specifically grafting a HCCA moiety on a phosphorylated position in order to amplify the signal of interest peptide.At the same time, we have developed a methodology for the study of ligand peptidic binding to its receptor which does not require radioactivity. The covalent HCCA tagged ligand has allowed us to detect and quantify it, in a binding displacement assay. More particularly, this methodology allowed us to determine the affinity of a reference ligand for the V1A vasopressin receptor.

As proteínas são consideradas importantes alvos para os oxidantes, devido à abundância em sistemas biológicos e às altas constantes de reações com estas espécies. Adicionalmente, têmse demonstrado que o triptofano (W) é um aminoácido extremamente susceptível a oxidação, inclusive pelo oxigênio singlete (1O2). A reação do W com o 1O2 tem despertado o interesse de diversos pesquisadores. Recentemente, esta reação tem atraído mais atenção, uma vez que produtos de oxidação do W tais como N-formilquinurenina (FMK) e quinurenina (kn) têm sido associados com algumas condições patológicas, tais como o desenvolvimento de catarata e a formação de agregados covalentes da superóxido dismutase envolvidos na esclerose lateral amiotrófica. Entretanto, há poucos trabalhos enfocando detalhadamente as reações, com estudos de estabilidade, identificação de subprodutos e propostas mecanísticas. Desta forma, pretendemos com este trabalho contribuir no esclarecimento do mecanismo de oxidação do triptofano pelo 1O2, através da análise e caracterização de produtos de oxidação gerados. Com este intuito, dois hidroperóxidos de triptofano isômeros (WOOH cis e trans, em relação ao grupamento carboxila) foram completamente caracterizados por análises de HPLC/espectrometria de massa e RMN como os principais produtos de oxidação do W pelo 1O2. Estes hidroperóxidos demonstraram ser relativamente estáveis às temperaturas ambiente e fisiológica, decompondo lentamente para os alcoóis correspondentes. O aumento do pH e/ou o aquecimento das soluções contendo os WOOH leva a decomposição quimiluminescente dos WOOH à FMK. Utilizando hidroperóxidos isotopicamente marcados com [18O] (W18O18OH) foi possível confirmar que a FMK formada durante esta decomposição era marcada com dois átomos de oxigênio. Este resultado demonstra que os dois átomos da FMK são derivados do grupamento hidroperóxido. Em adição, estas reações são quimiluminescentes, sugerindo o envolvimento de um intermediário dioxetano. Este mecanismo foi confirmado uma vez que o espectro de quimiluminescência da decomposição dos WOOH pode ser sobreposto ao espectro de fluorescência da FMK, inequivocamente identificado a FMK como a espécie emissora. Dioxindoilalaninas diastereoisoméricas também foram caracterizadas como produtos de oxidação do triptofano pelo 102; uma possível via radicalar foi excluída. Em suma, este estudo contribuiu na elucidação das bases químicas envolvidas na oxidação do triptofano por 102, através da caracterização dos produtos formados e da investigação detalhada dos mecanismos de decomposição destes produtos. / Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen (1O2). The reaction of 1O2 with W has long been a matter of concern, and has recently attracted considerably more attention because W-derived oxidation products such as N-formylkynurenine (FMK) and kynurenine (kn) have been associated with some pathological conditions such as the development of cataracts and the formation of covalent aggregates of superoxide dismutase, which has been implicated in amyotrophic lateral sclerosis. Despite the intense interest in the mechanism of W oxidation, there are a lot of gaps that remains to be elucidated. In this context, the current study was undertaken to investigate the chemical basis involved in W oxidation by 1O2. We are concerned about the chemistry of the initially formed hydroperoxides, their stability, further reactions and the mechanism leading to FMK conversion. With this purpose, two cis and trans tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. Also, they were shown to be relatively stable at ambient and physiological temperatures, leading to a slow decomposition to the corresponding alcohols. Increasing the pH or heating the solutions gives rise to a luminescent decomposition of the WOOH to FMK. Using 18O-labeled hydroperoxides (W18O18OH), it was possible to confirm the formation of a two-oxygen-labeled FMK molecule derived from W18O18OH decomposition. This result shows that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species. Diastereoisomeric dioxindoyalanine were also characterized as oxidation products derived from the reaction of W with 1O2. The involvement of radicals in this reaction was excluded. In summary, this work offers further insights into the chemistry involved in W-oxidation, through the characterization of photoproducts and the detailed investigation about the decomposition mechanism of these products.

INTRODUÇÃO: Apesar dos Inibidores Seletivos da Recaptura da Serotonina (ISRSs) serem a primeira escolha no tratamento do Transtorno de Estresse Pós-Traumático (TEPT) seu mecanismo de ação não é completamente compreendido. Possivelmente, um aumento na resiliência ao estresse esteja envolvido. Como a serotonina (5HT) ajuda a mediar as respostas ao estresse em outros transtornos ansiosos, o paradigma de Depleção de Triptofano Aguda (DTa) foi usado para diminuir a 5HT central em Pacientes com TEPT remitido com ISRSs. MÉTODOS: Dez pacientes com TEPT (diagnosticados pela Mini Entrevista Neuropsiquiátrica Internacional) que tiveram resposta completa com um ISRS (Escala de Impressões Clínicas Globais de Melhora 1-2 por pelo menos 3 meses) foram selecionados para o experimento. Os pacientes foram testados em duas ocasiões diferentes separadas por uma semana nas quais os pacientes receberam uma mistura contendo grandes aminoácidos neutros ou com (Depleção de Triptofano Falsa [DTf]; dia controle) ou sem triptofano (dia DTa). Auto relatos de ansiedade e humor, bem como medidas cardiovasculares, foram obtidos ao longo dos testes. Cinco horas e meia após a ingestão da mistura os pacientes foram re-expostos aos seus traumas através do procedimento de imaginação guiada de Pitman. RESLTADOS: Esses procedimentos provocaram elevados escores nas medidas avaliadas em ambos os dias, com respostas significativamente mais intensas no dia DTa conforme avaliado pelas Escalas Visuais Analógicas (DTa 47,57 [21,75] -v- DTf 20,71 [18,4]; p=0,001), Escala de Trauma de Davidson (29,4 [12,7] -v- 15,7 [7,79]; p=0,001), Inventário de Ansiedade de Spielberger versão Estado (28,9 [11,03] -v- 18,5 [10,13]; p=0,066, e Perfis de Estados de Humor (p<0,001). CONCLUSÕES: Esses dados são os primeiros a demonstrar que a depleção de 5HT piora as respostas subjetivas a re-experimentação de memórias traumáticas no TEPT e sugere que o aumento na função da 5HT induzida por ISRSs diminui os sintomas de TEPT, especialmente sob provocação, i.e. 5HT ajuda a mediar a resiliência ao estresse. Além de fornecer insights sobre o como os ISRSs funcionam no TEPT, esses dados também oferecem uma abordagem de potenciais novos tratamentos para esse transtorno / INTRODUCTION: Although Selective Serotonin Reuptake Inhibitors (SSRIs) are the first-line Posttraumatic Stress Disorder (PTSD) treatments their mechanism of action is unclear, but possibly improvement of stress resilience is involved. As serotonin (5HT) helps regulate stress responses in other anxiety disorders, the acute tryptophan depletion (aTD) technique was used to lower brain 5HT in SSRSs-remitted PTSD patients. METHODS: Ten patients with PTSD (Mini-International Neuropsychiatric Interview diagnosed) who had made a full recovery on SSRIs (Clinical Global Impressions Improvement Scale 1-2 for at least 3 months) were enrolled in the experiment. Patients were tested on 2 separate occasions a week apart - each session they received a drink containing large neutral amino acids either with (Sham Depletion [SD]; control day) or no tryptophan (aTD day). Self reports of anxiety and mood, as well as cardiovascular measures, were obtained throughout the tests. At 5.5 hours after the drink subjects were reexposed to their trauma using a modification of Pitmans imagery guided method. RESULTS: These procedures provoked elevated ratings on both days, with significantly more marked responses on the aTD day according to Visual Analogue Scales (aTD 47.57 [21.75] -v- SD 20.71 [18.4]; p=0.006), Davidson Trauma Scale (29.4 [12.7] -v- 15.7 [7.79]; p=0.001), Spielberger State Anxiety Inventory (28.9 [11.03] -v- 18.5 [10.13]; p=0.066, and Profile of Mood States (p<0.001). CONCLUSIONS: These data are the first to show that 5HT depletion worsens the subjective responses to reliving traumatic memories in PTSD and suggest that that SSRI-induced increases in 5HT function restrains PTSD symptoms, especially under provocation, i.e. 5HT helps mediate resilience to stress. As well as giving insights into how SSRIs work in PTSD, these data may also offer a translational approach to potential new treatments for this disorder

A glândula pineal de mamíferos sintetiza o hormônio melatonina exclusivamente durante o período noturno. A síntese é regulada primordialmente pela via retino-hipotalâmico-pineal e modulada por vários fatores, incluindo o sistema peptidérgico. Assim, o papel da insulina na regulação da síntese de melatonina foi estudado a partir da realização de culturas de glândulas pineais estimuladas com noradrenalina, insulina e noradrenalina associada à insulina, em culturas temporizadas ou não pela noradrenalina, avaliando: a produção de melatonina por HPLC com detecção eletroquímica; as atividades das enzimas envolvidas na síntese da melatonina, por radiometria; assim como, a expressão gênica das enzimas quantificada por Real-Time PCR. Os resultados sugerem uma interação entre as vias de sinalização da noradrenalina e da insulina, com a respectiva potencialização da síntese da melatonina, induzida por noradrenalina, observada pela adição da insulina, efeito esse, que se dá, provavelmente através de mecanismos pós-transcricionais. / The mammalian pineal gland synthesizes the neurohormone melatonin exclusively during the dark phase. Its synthesis is primarily regulated via a retino-hypothalamic-pineal pathway and modulated by many factors, including the peptidergic system. Thus, the role of insulin on the regulation of melatonin synthesis was studied using cultured gland treated with norepinephrine, insulin and norepinephrine associated to insulin. The cultures were also synchronized or not by norepinephrine. Melatonin content was assayed by HPLC (High Performance Liquid Chromatography) with electrochemical detection, melatonin synthesis enzymes activities by radiometry and enzymes gene expressions by Real-Time PCR. The results suggest an interaction between norepinephrine and insulin signaling pathway, with insulinic potentialization on melatonin synthesis norepinephrine-mediated, and this effect, seems to accurs potentially through post-transcriptional events.

Zielbereiche der vorliegenden Arbeit stellten sowohl die Analysen zur Auswirkung eines Schlafentzuges (SE) unter Tryptophan-Depletion (TD) auf die hormonellen Parameter Prolaktin (PRL), Cortisol, Thyreotropin (TSH), Trijodthyronin (T3) und Thyroxin (T4) als auch die daraus resultierenden Rückschlüsse im Hinblick auf eine Verzahnung dieser Parameter nicht nur mit der depressiven Symptomatik sondern auch mit den SE-Wirkmechanismen dar. Zur Untersuchung dieser bislang im Rahmen der biologischen Depressionsforschung unberücksichtigten Kombination gelangten 22 SE-Responder, die sich einem doppelblind aktiv placebokontrollierten (SHAM) Parallelgruppendesign unterzogen. Die eigenen Beobachtungen einer blanden Response nach SE unter TD im Vergleich zur SHAM-Version des PRL und TSH einerseits sowie des Cortisols andererseits im Sinne eines abgeschwächten Plasmakonzentrationsanstiegs bzw. -abfalls sprechen zugunsten der Hypothese, daß der SE über serotonerge Wirkmechanismen zumindest teilreguliert werden dürfte. Die Konzentrationsunterschiede zwischen den Untersuchungsgruppen, die am SE-Nachmittag für PRL, Cortisol und TSH signifikant ausfallen, trotz gleichzeitig deutlicher Besserung der depressiven Symptomatik aller Patienten, legen zudem die Folgerung nahe, daß die divergierende hormonelle Antwort auf eine TD als Trigger einer klinischen Differenzierbarkeit der Depressionstiefe insuffizient zu sein scheint. / The present double-blind and placebo-controlled study was designed to analyse the impact of sleep-deprivation under tryptophane-depletion in depressed patients on hormones, e.g. prolactine, cortisol, TSH, T3, and T4. Current data support the hypothesis that sleep-deprivation might act as an antidepressant by serotonergic pathways. Thus, alterations of these relevant humoral parameters could expected by decreased serotonergic activity after tryptophane-depletion. We investigated 22 depressed patients who responded to one night of sleep-deprivation. Tryptophane-depletion led to a blunted increase of prolactine and TSH, whereas cortisol plasma-level revealed a blunted decrease. No difference between tryptophane-depletion and placebo could be spot concerning neither T3 nor T4. Our results support the idea that serotonergic pathways might be involved in the interactions following sleep-deprivation, but cannot explain the complex alterations of the aforementioned hormones alone.