Atrial fibrillation (AF), the most common arrhythmia encountered in clinical practice, is a major source of morbidity and mortality, and is highly associated with inflammation and oxidative stress. In the present study, we show that acute exposure of mice atrial tissue to tumor necrosis factor-α (TNF-α) increases susceptibility to AF. We further show that acute exposure to TNF-α led to increased spontaneous sarcoplasmic reticulum (SR) calcium release and generated triggered activities in isolated mice atrial myocytes. This increase in spontaneous SR calcium activity was found to be due to elevated reactive oxygen species production from mitochondria and NADPH oxidase sources triggered by TNF-α. Hence we concluded that acute exposure to TNF-α leads to elevated oxidative stress that increases spontaneous SR Ca2+ release and triggered activity through which it can lead to AF induction and maintenance

Atrial fibrillation (AF), the most common arrhythmia encountered in clinical practice, is a major source of morbidity and mortality, and is highly associated with inflammation and oxidative stress. In the present study, we show that acute exposure of mice atrial tissue to tumor necrosis factor-α (TNF-α) increases susceptibility to AF. We further show that acute exposure to TNF-α led to increased spontaneous sarcoplasmic reticulum (SR) calcium release and generated triggered activities in isolated mice atrial myocytes. This increase in spontaneous SR calcium activity was found to be due to elevated reactive oxygen species production from mitochondria and NADPH oxidase sources triggered by TNF-α. Hence we concluded that acute exposure to TNF-α leads to elevated oxidative stress that increases spontaneous SR Ca2+ release and triggered activity through which it can lead to AF induction and maintenance

Combinatorial protein engineering, taking advantage of large libraries of protein variants and powerful selection technology, is a useful strategy for developing affinity proteins for applications in biotechnology and medicine. In this thesis, two small affinity proteins have been subjected to combinatorial protein engineering to improve or redirect the binding. In two of the projects, a three-helix protein domain based on staphylococcal protein A has been used as scaffold to generate so called Affibody molecules capable of binding to key proteins related to two diseases common among elderly people. In the first project, Affibody molecules were selected using phage display technology for binding to Ab-peptides, believed to play a crucial role in Alzheimer’s disease, in that they can oligomerize and contribute to the formation of neural plaques in the brain. The selected Affibody molecules were found to efficiently capture Ab from spiked human plasma when coupled to an affinity resin. The structure of the complex was determined by nuclear magnetic resonance (NMR) and demonstrated that the original helix 1 in the two Affibody molecules was unfolded upon binding, forming intermolecular b-sheets that stabilized the Ab peptide as buried in a tunnel-like cavity. Interestingly, the complex structure also revealed that the Affibody molecules were found to homo-dimerize via a disulfide bridge and bind monomeric Ab-peptide with a 2:1 stoichiometry. Furthermore, Affibody molecule-mediated inhibition of Ab fibrillation in vitro, suggested a potential of selected binders for future therapeutic applications. In the second project, two different selection systems were used to isolate Affibody molecules binding to tumor necrosis factor alpha (TNF), which is involved in inflammatory diseases such as rheumatoid arthritis. Both selection systems, phage display and Gram-positive bacterial display, could successfully generate TNF-binding molecules, with equilibrium dissociation constants (KD) in the picomolar to nanomolar range. Initial characterization of the binding to TNF was evaluated by competitive binding studies between the Affibody molecules and clinically approved TNF antagonists (adaliumumab, infliximab and etanercept) and demonstrated overlapping binding sites with both adaliumumab and etanercept. Furthermore, linkers of different lengths were introduced between Affibody moieties, in dimeric and trimeric constructs that were evaluated for their ability to block the binding between TNF and a recombinant form of its receptor. In the dimeric constructs, a linker length of 20-40 amino acids seemed to have an advantage compared to shorter and longer linkers, and the tested trimeric construct could block the TNF binding at even lower concentration. The results provided valuable information for the design of future Affibody-based molecules that could be investigated in therapeutic or medical imaging applications. In the third project aiming to generate a protein domain with capacity to influence the pharmacokinetics of protein therapeutics, a natural serum albumin-binding domain (ABD) was subjected to an engineering effort aiming at improving the affinity to human serum albumin (HSA), a protein with an exceptional long half-life in serum (19 days). First-generation affinity improved ABD variants were selected using phage display technology from a constructed ABD library. After additional rational engineering of such first generation variants, one variant with a 10,000-fold improved affinity to HSA (KD ≈ 120 fM) was obtained. Furthermore, characterization of this molecule also demonstrated improved affinity to several other serum albumins. When used as a gene fusion partner, this affinity-maturated variant denoted ABD035, should have the potential to extend the half-life of biopharmaceuticals in humans, and several other animal species. / QC 20100722

La apoptosis es un proceso de muerte celular programada que está involucrada en la selección del repertorio de linfocitos T y en el mantenimiento de la tolerancia inmunológica, ya que es el mecanismo por el que se eliminan las células que podrían dar lugar a respuestas autoinmunes. Existen evidencias de que la alteración en los mecanismos apoptóticos están implicados en la patogenia y la actividad de las enfermedades autoinmunes sistémicas, de las cuales, el lupus eritematoso sistémico (LES) es la más representativa. Se han identificado en el ser humano algunos genes que codifican oncoproteínas y citocinas cuya transcripción parece ser crucial en este proceso: unos pro-apoptóticos (fas, p53 y TNF-alfa) y otros anti-apoptóticos (bcl-2 y IL-10). Se sospecha que cambios en la expresión de los mismos podrían desempeñar algún papel en la patogenia del LES, al favorecer la proliferación de determinadas poblaciones celulares de efecto autorreactivo.Con los trabajos de la presente tesis doctoral nos propusimos: determinar la implicación de los oncogenes (bcl-2, fas y p53) y las citocinas (IL-10 y TNF-alfa) en la disregulación apoptótica que presentan los pacientes con LES; estudiar la interrelación existente entre ellos; y analizar la relación de las posibles disregulaciones de los elementos que participan en la apoptosis con la actividad de la enfermedad lúpica.Los resultados obtenidos muestran cómo los oncogenes fas, bcl-2 y p53, las citocinas IL-10 y TNF-alfa, y la fracción proteica soluble del Fas (sFas) tienen una notable importancia en la patogenia y la actividad de la enfermedad lúpìca. Las vías apoptóticas del Fas y p53 son independientes entre sí. Sin embargo, diversas citocinas (IL-10, TNF-alfa), oncoproteínas (Bcl-2) y fracciones proteicas solubles (sFas) pueden ser las encargadas de relacionarlas entre sí. La interferencia de estas vías apoptóticas produciría una eliminación deficiente de los linfocitos autorreactivos. Ello favorecería su supervivencia, lo que provocaría las alteraciones de disregulación inmunológica propias del LES.

Osteoclasts are multinucleated cells found exclusively in bone and are derived from the haematopoietic cells of monocytes/macrophage lineage. The cell-to-cell interaction between osteoblastic/stromal cells and osteoclast precursor cells is necessary for osteoclastogenesis. Receptor Activator of NF-κB ligand (RANKL) was identified as a membrane-bound TNF ligand family member that is the ‘master’ cytokine expressed on osteoblastic/stromal cells, which stimulate osteoclastogenesis through cell-to-cell contact with osteoclast precursors. RANKL is considered to be a factor that is necessary and sufficient for the induction of osteoclastogenesis (Lacey, et al., 1998). RANKL is a type II transmembrane cytokine of the TNF ligand superfamily and has an active TNF-like core domain at the extracellular domain. This active TNF-like core domain is thought to be the region through which it binds to it’s active receptor, RANK, for the activation of signal transduction pathways for the initiation of processes leading to osteoclastogenesis (Lacey, et al., 1998; Li, et al., 1999). It was hypothesized that any change in the active TNF-like core domain might affect the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. Hence, this thesis sought to investigate the effects of changes in the active TNF-like core domain by truncation mutation on the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. A cDNA fragment encoding the full-length TNF-like core domain of rat RANKL (rRANKL) (aa160-318) was cloned into the bacterial expression pGEX vectors and stably expressed in Eschechia coli as a fusion protein with the C-terminus of glutathione S-transferase (GST). Four mutants (aa160-302, aa160-268, aa239-318 and aa246-318) were also generated by truncation mutation in the TNF-like core domain, and cloned into the pGEX vector to produce GST-rRANKL mutants. The proteins were over-expressed and affinity purified to 95% in purity. GST-rRANKL (160-318) containing the full length TNF-like core domain was able to induced osteoclastogenesis in spleen cells in the presence of M-CSF and in RAW264.7 cells in the absence of M-CSF. It was also found to activate mature osteoclast activity in vitro, ex vivo and in vivo. It has the highest binding affinity to RANK and the greatest potency for NF-κB activation as well as the induction of osteoclastogenesis compared to the truncated mutants. Mutants generated by truncation of the TNF-like core domain revealed that the TNF-like core domain is important for the interaction with the RANK, for high binding affinity, NF-κB activation and induction of osteoclastogenesis. In general, the truncated mutants not only displayed a reduction in the binding affinity to RANK, but also a reduction in NF-κB activation, and significantly reduced potency in the induction of osteoclastogenesis. Interestingly, mutant GST-rRANKL (160-268) showed a higher affectivity than the other mutants did, in that it had greater binding affinity to RANK, and in NF-κB activation than the rest of the truncated mutants. Mutants GST-rRANKL (239-318) and GST-rRANKL (246-318) on the other hand, showed little potency in the induction of osteoclast formation, however, might have an inhibitory effect through competition with full length GST-rRANKL (160-318) as well as inducing a response in vivo resulting in an increase in the serum calcium level. In conclusion, this thesis demonstrated that the TNF-like core domain of RANKL is active, and imperative in the binding to RANK, activating signal transduction pathways and induction of osteoclastogenesis. Changes in the active TNF-like core domain affected the ability, affinity and efficiency of RANKL binding to the receptor, RANK and consequently affected the activation of signal transduction pathways and osteoclastogenesis.

Ο λιπώδης ιστός είναι πλέον ένα ενδοκρινές όργανο το οποίο εκκρίνει ένα μεγάλο αριθμό βιοδραστικών μεσολαβητών που στοχεύουν σε όργανα μεταβολικής σημασίας όπως ο εγκέφαλος, το ήπαρ, οι σκελετικοί μύες και το ανοσοποιητικό σύστημα, ρυθμίζοντας την ομοιόσταση, την αρτηριακή πίεση, το μεταβολισμό των λιπιδίων και της γλυκόζης, την φλεγμονή και την αθηροσκλήρυνση. Η αντιπονεκτίνη κύρια πρωτεΐνη παραγόμενη από τον λιπώδη ιστό δρα προστατευτικά στην ανάπτυξη ινσουλινοαντίστασης, φλεγμονής και αθηρωμάτωσης. Η παχυσαρκία και κυρίως η κεντρική παχυσαρκία συνδέεται με την εμφάνιση πολλών νοσογόνων καταστάσεων. Στο σπλαχνικό λίπος εκκρίνεται TNF-α από τα λιποκύτταρα και τα μακροφάγα του στρώματος, ο οποίος καταστέλλει την παραγωγή και την δράση της αντιπονεκτίνης δρώντας ευωδοτικά στην ανάπτυξη του μεταβολικού συνδρόμου. Μελετήσαμε τις διαφορές της έκφρασης της αντιπονεκτίνης και του TNF-α σε σπλαχνικό και υποδόριο λιπώδη ιστό με τη μέθοδο της έμμεσης ανοσοϊστοχημείας σε τρεις ομάδες ασθενών: υγιείς, παχύσαρκους υγιείς και σε παχύσαρκους με μεταβολικό σύνδρομο και Σ.Δ. / Adipose tissue, besides of its energy producting role, is now considered as an endocrine organ which releases numerous cytokines and adipokines. Adipose tissue crosstalks with immune, cardiovascular reproductive and other systems. In obesity (BMI>30) the adipocytes become hypertrophic, loosing their normal activity and increasing the risk of appearence of the Metabolic Syndrome (a cluster of risk factors which may lead to Coronary Artery Disease). Adiponectin is an adipokine which plays pivotal role protecting from Diabetes Melitus t. 2 and inflammation. In obesity, the level of serrum adiponektin is remarkably low. TNF-α is a multifunctional cytokine released mostly from the immune cells. Central obesity (waist circumference >102cm) is characterized of a low grade inflammation combined with decreased secretion of TNF-α from adipocytes and stroma cells of visceral adipose tissue. In central obesity TNF-α acts against insulin leading to the appearance of insulin resistance and M.S. In our study we examined the different levels of secretion of adiponectin and TNF-α from the adipocytes of subcutaneous and visceral fat between three categories of patients: a) Controls, b) Obese without central obesity M.S. and c) Obese with M.S.

Autoimmune diseases are a group of different diseases which are characterized by an immune disorder leading to decreased tolerance to components of the body itself. These diseases have many factors that trigger such as a decrease of the share or percentage of regulatory T cells (Tregs). The Transforming Growth Factor-beta 1 (TGF-β1) is involved in the suppression of the inflammatory response during the pathogenesis of autoimmune diseases (juvenile idiopathic arthritis, multiple sclerosis, diabetes), through the activation of this cell type. This cytokine is also associated with modulation of an inflammatory response either by increasing Treg cells and by modulating proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α). The components found in both innate immune responses as adaptive must be considered potential targets for developing new drugs immune modulators. Thus, manipulation of Tregs is an attractive strategy for immunotherapy and hence, the use of mimetic peptide to TGF-β1 can be adopted to reduce the effects of severe autologous response, then creating an additional therapy for autoimmunity as well as for the treatment of inflammatory diseases. Our results show that we can select TGF-β1 mimetic peptides since we can prove by bioinformatics both bind to this receptor molecule. Thus, the peptides can be used as immunomodulators to combat inflammation and in the treatment of autoimmune diseases since they can modulate the production of TNF-α and IL-10. / Doenças autoimunes são um grupo de doenças distintas que se caracterizam por uma desordem imunológica levando a diminuição da tolerância aos componentes do próprio organismo. Essas doenças possuem vários fatores que as desencadeiam como a diminuição da ação ou porcentagem de células T regulatórias (Tregs). O Fator Transformante de Crescimento-beta 1 (TGF-β1) está envolvido na supressão da resposta inflamatória durante a patogênese de doenças autoimunes (artrite idiopática juvenil, esclerose múltipla, diabetes), por meio da ativação desse tipo celular. Esta citocina também está associada a modulação de uma resposta inflamatória, seja pelo aumento de células Tregs como pela modulação de citocinas pro-inflamatórias como o Fator de necrose tumoral alfa (TNF-α). Os componentes encontrados em respostas tanto imune inatas quanto adaptativas devem ser considerados potenciais alvos para o desenvolvimento de novos fármacos imuno moduladores. Assim, a manipulação de Tregs é uma estratégia atraente para a imunoterapia e, desta forma, o uso de peptídeos miméticos ao TGF-β1 poderá ser adotado para diminuir as consequências de uma resposta autóloga severa, criando, então, uma terapia complementar para a autoimunidade bem como para o tratamento de doenças inflamatórias. Nossos resultados mostram que conseguimos selecionar peptídeos miméticos a molécula do TGF- β1, uma vez que conseguimos provar por bioinformática que ambos se ligam ao receptor desta molécula. Assim, os peptídeos podem ser utilizados como imunomoduladores para o combate de inflamação e no tratamento de doenças autoimune já que conseguem modular a produção de TNF- α e IL-10. Experimentos in vivo realizados também demonstraram a sua capacidade de diminuir inflamação modulação a migração de neutrófilos e leucócitos. / Mestre em Genética e Bioquímica

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-18T11:57:11Z No. of bitstreams: 2 camila_danielle_cunha_neves.pdf: 1114741 bytes, checksum: d50be0f18b6b46636a9e4cf8ef159a66 (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-18T12:22:44Z (GMT) No. of bitstreams: 2 camila_danielle_cunha_neves.pdf: 1114741 bytes, checksum: d50be0f18b6b46636a9e4cf8ef159a66 (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-18T12:22:41Z (GMT) No. of bitstreams: 2 camila_danielle_cunha_neves.pdf: 1114741 bytes, checksum: d50be0f18b6b46636a9e4cf8ef159a66 (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-18T12:23:34Z (GMT) No. of bitstreams: 2 camila_danielle_cunha_neves.pdf: 1114741 bytes, checksum: d50be0f18b6b46636a9e4cf8ef159a66 (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Made available in DSpace on 2014-12-18T12:23:37Z (GMT). No. of bitstreams: 2 camila_danielle_cunha_neves.pdf: 1114741 bytes, checksum: d50be0f18b6b46636a9e4cf8ef159a66 (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Previous issue date: 2014 / O efeito do consumo cr?nico do cigarro no perfil inflamat?rio, estresse oxidativo e desempenho f?sico t?m sido investigado em fumantes assintom?ticos, contudo, estes estudos na sua maioria ainda s?o poucos e divergentes. Sendo assim, o objetivo deste estudo foi avaliar o efeito cr?nico do tabagismo no perfil inflamat?rio, estresse oxidativo, fun??o muscular esquel?tica perif?rica e aptid?o cardiorrespirat?ria de homens adultos assintom?ticos. Os sujeitos do estudo foram divididos igualmente entre o grupo fumante (GF, n= 20) e grupo controle (GC, n= 20), composto por sujeitos n?o fumantes. A fun??o pulmonar de todos participantes foi avaliada pela espirometria. O perfil inflamat?rio foi avaliado pela mensura??o das concentra??es plasm?ticas das citocinas IL-6, TNF-?, IL-10 e pelos receptores sol?veis de TNF-? (sTNFR1 e sTNFR2). O estresse oxidativo foi avaliado pela mensura??o das concentra??es plasm?ticas das subst?ncias reativas ao ?cido tiobarbit?rico (TBARS), da atividade das enzimas antioxidantes super?xido dismutase (SOD) e catalase de eritr?citos e pela capacidade antioxidante total do plasma. A fun??o muscular esquel?tica perif?rica foi avaliada por meio das medidas de pico de torque dos m?sculos extensores e flexores de joelho e da resist?ncia e fadiga muscular dos m?sculos extensores de joelho; e a aptid?o cardiorrespirat?ria foi avaliada atrav?s da mensura??o do consumo pico de oxig?nio (VO2pico), frequ?ncia card?aca m?xima (FCmax) e dist?ncia caminhada em um teste m?ximo de caminhada/corrida. Sujeitos do GF apresentaram aumentos significativos das concentra??es plasm?ticas de TBARS e do receptor sol?vel sTNFR1 e diminui??es significativas da atividade da enzima catalase em lisado eritrocit?rio, comparados ao GC. Al?m disso, sujeitos do GF exibiram menor resist?ncia muscular dos m?sculos extensores de joelho como determinado pelas medidas de trabalho, com inalterado ?ndice de fadiga dos m?sculos extensores de joelho e pico de torque dos m?sculos extensores e flexores de joelho. A aptid?o cardiorrespirat?ria foi similar entre os sujeitos do GF e do GC. Este estudo demonstrou que homens fumantes cr?nicos e assintom?ticos exibiram altera??es no perfil inflamat?rio, no estresse oxidativo e na fun??o muscular esquel?tica perif?rica, quando comparados a n?o fumantes. / The effect of chronic cigarette smoking on inflammatory profile, oxidative stress and physical performance has been investigated in asymptomatic smokers, however, these studies are few and divergent. Thus, the aim of this study was to evaluate the chronic effect of smoking on inflammatory profile, oxidative stress, peripheral skeletal muscle function and cardiorespiratory fitness in asymptomatic adult men. The subjects of the study were divided equally between the smoking group (SG, n = 20) and control group (CG, n = 20) composed of non-smoking subjects. The lung function of all participants was assessed by spirometry. The inflammatory response was assessed by measurement of plasma concentrations of IL- 6, TNF- ?, IL -10 and soluble receptors for TNF-? (sTNFR1 and sTNFR2). Oxidative stress was evaluated by measuring plasma concentrations of thiobarbituric acid reactive substances (TBARS), the activity of antioxidant enzymes superoxide dismutase (SOD) and catalase in erythrocytes and the total antioxidant capacity of plasma. The peripheral skeletal muscle function was assessed by measuring the peak torque of the extensor and flexor muscles of the knee; and resistance and muscle fatigue of the knee extensor muscles; and cardiorespiratory fitness was assessed by measurement of peak uptake oxygen (VO2peak), maximum heart rate (HRmax) and distance walked on a maximum test of the walk/run. SG subjects showed significant increases in plasma concentrations of TBARS and soluble receptors sTNFR1 and significant decreases in erythrocyte catalase enzyme activity, compared to the CG. In addition, SG subjects exhibited less muscle strength of knee extensors muscles as determined by measures of work and unchanged fatigue index of the knee extensors muscles and peak torque of knee extensors and flexors muscles. Cardiorespiratory fitness was similar between subjects SG and CG. This study demonstrated that male chronic smokers, healthy adult, exhibited changes in the inflammatory response, oxidative stress and peripheral skeletal muscle function, when compared to non-smokers. / Disserta??o (Mestrado) ? Programa Multic?ntrico de P?s-gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2014.

Introdução: a resposta neuro-endócrina metabólica perioperatória está associada ao estresse, fadiga, ansiedade e dor. No entanto, poucos estudos têm investigado a interação entre os sistemas neuro-imuno-endócrino com o estresse pré-operatório e a recuperação pós-cirúrgica. Um aspecto relevante nesta questão é o envolvimento do sistema neuroimuno- endócrino na relação entre estresse e dor pós-operatória. Objetivo: o presente estudo avaliou os níveis diurnos e noturnos da melatonina, do TNF e do cortisol em mulheres submetidas a histerectomia nos períodos pré e pós-operatórios com intuito de avaliar a regulação da síntese de melatonina pela glândula pineal por meio das vias da inflamação e neuroendócrina. Métodos e resultados: avaliou-se 12 pacientes submetidas a histerectomia abdominal. As amostras de sangue foram coletadas às 10h00 e 22h00, uma semana antes da hospitalização, no dia prévio à cirurgia, 1O e o 2O dias de pós-operatório e às 22h00 do dia da cirurgia. Na noite da cirurgia não houve níveis mensuráveis de melatonina noturna. Foram utilizados questionários para avaliar os níveis de estresse e dor e a relação da intensidade destes sintomas clínicos e o perfil bioquímico. Usando um modelo de Equação de Estimativa Generalizada [Generalized Estimating Equations (GEE)], evidenciou-se que os níveis séricos aumentados do TNF e do cortisol, bem como o estado de ansiedade elevado suprime a secreção da melatonina noturna. Para analisar a relação entre a secreção da melatonina ao longo do tempo e sua relação com cada um dos seguintes fatores: TNF, cortisol sérico matinal e estado de ansiedade, em primeiro lugar, estes fatores foram categorizados tendo como referencial os quartis. De acordo com cada ponto de corte as médias e os desvios padrões da melatonina noturna foram: TNF (alto > Q75=5.2 vs. baixo Q75 < 5.2 ng/mL) 57,35 + 40,85 vs 24,17 + 11,79 (P= 0.04); cortisol sérico matinal (alto > Q50=22.4 vs. baixo Q50 < 22.4 ng/mL) 33,29 + 6,29 vs 14,41 + 2,19 (P= 0.03) e ansiedade estado (alta > Q50=24 vs. baixa Q50 < 24 pontos no escore) 43,66 + 8,55 vs 31 + 6,46 (P= 0.03), respectivamente. Além disso, maiores níveis de dor pós-operatória na escala analógica visual (VAS), bem como maior ansiedade-traço se relacionaram com menores níveis de melatonina noturna perioperatória. Conclusão: os resultados deste trabalho reforçam a hipótese de que a glândula pineal desempenha um papel expressivo na reação ao estresse cirúrgico. A elevação dos níveis de TNF na fase pró-inflamatória, e do cortisol suprimem a produção da melatonina pela pineal, desligando o braço hormonal, o que permite a montagem da resposta de defesa. Considerando tais evidências, a reação que ocorre entre o TNF, o cortisol e a glândula pineal é essencial para permitir uma recuperação completa e eficiente no período pósoperatório. / Background: Neuro-endocrine metabolic response in the perioperative period is associated with distress, fatigue, anxiety, and acute postoperative pain. However, few researches have investigated the interaction between the neuroendocrine-immune system, preoperative stress, and post-surgical recovery. A relevant aspect in this equation is the involvement of the neuroendocrine-immune interaction in the relationship between preoperative stress and post-surgical pain. Objective: The present study followed the daytime and nighttime levels of melatonin, TNF an cortisol, in women that had undergone a histerechtomy from the pre-surgical to postsurgical periods out to study the regulation of melatonin synthesis by the pineal gland by airway inflammation and neuroendocrine. Methods and results: We evaluated 12 women who underwent an abdominal hysterectomy. Blood was collected, at 10h00 and 22h00, one week and one day before surgery; at the first and second days after surgery and at 22h00 on the day of surgery. On the night of surgery there was no melatonin surge. In addition questionnaires for evaluating stress and pain levels showed a correlation between personal information and biochemical profiles. Generalized Estimating Equations (GEE) revealed that high TNF and serum cortisol, as well high state-anxiety decrease the nocturnal melatonin surge. To analyze the relationship between the melatonin surge on time and its relationship with each one of the following factors, firstly, they were classified according to cutoff points using quartiles. The mean and standard deviation of nocturnal melatonin was TNF (high > Q75=5.2 vs. low Q75 < 5.2 ng/mL) 57.35+ 40.85 vs 24.17+ 11.79, (P=0.04); morning serum cortisol (high > Q50=22.4 vs. low Q50 < 22.4 ng/mL) 33.29+ 6.69 vs. 14.41+ 2.19, (P=0.03) and state-anxiety (high > Q50=24 vs. low Q50 < 24 points], 43.66+ 8.55 vs. 31+ 6.46, (P=0.03); respectively. Also, the increase of postoperative pain on visual analogue scale (VAS), as well as higher trait-anxiety was linked with lower nocturnal melatonin surge in perioperative period. Conclusion: the results support the hypothesis that the pineal gland plays a role in the progressive reaction to surgical stress. In the proinflammatory phase, TNF shutdown melatonin production and allows for the mounting of the defense response. Also, it is observed that the cortisol is a determinant in the regulation of nocturnal melatonin surge, together with the release of high amounts of TNF in the circulation, at the start of a defense response, suppressing the melatonin surge. Taking into account these evidences, it is possible to suppose that this orchestrated reaction between TNF, cortisol, and the pineal gland should be essential in allowing for a full and efficient postoperative recovery.

Base teórica: A dor crônica decorre de alterações estruturais e funcionais mal adaptativas que influenciam a resposta ao estímulo ou que sustentam os processos de excitabilidade. Um dos sintomas que permeia grande número de pacientes com dor crônica é a catastrofização, cujas características são um conjunto de pensamentos negativos, deseperança e magnificação do sintoma ou condição. Este sintoma é mensurado por meio de uma escala de catastrofização usada em vários países. No entanto não dispomos deste instrumento validado para o português.Objetivos: Validar para o português do Brasil (B) a PCS e verificar suas propriedades psicométricas. Verificar a consistência interna, estrutura fatorial, e sua capacidade de discriminar pacientes com condições específicas de dor crônica como cefaleia tensional crônica (CTC) (International Headache Society) e fibromialgia de acordo com os critérios do American College of Rheumatology. Avaliar os possíveis mecanismos neurobiológicos correlacionados com o nível de sintomas catastróficos, através de dosagens de cortisol e TNF em uma amostra de pacientes com CTC. Métodos: 384 sujeitos com idades entre 18-79 anos com dor crônica de origem músculo-esquelética participaram deste estudo transversal. A versão da B-PCS foi aplicada, assim como a intensidade da dor, interferência da dor na capacidade funcional, no humor e um questionário sócio-demográfico. A capacidade discriminatória da B-PCS foi avaliada numa sub-amostra de pacientes com cefaléia tensional crônica (CTC) de acordo com os critérios da International Headache Society (n = 19), e em outro com diagnóstico de fibromialgia segundo os critérios do American College of Rheumatology (n = 50). Após a validação a B-PCS foi aplicada num grupo de pacientes com CTC. Foi avaliado o impacto da cefaleia usando o Short-Form Headache Impact Test (HIT-6), coletadas amostras de cortisol salivar às 08:00; 16:00 e 22:00 e dosado o TNF sérico. Resultados: Observou-se boa consistência interna [valores α de Cronbach de 0,91 para o total da BR-PCS. Para os subdomínios 0,93 (desesperança), 0,88 (magnificação), 0,86 (ruminação)]. Os coeficientes de correlação item-total variaram 0,91-0,94. Análise fatorial confirmatória apoiou os três fatores de estrutura, com o índice de ajuste comparativo = 0,98, a raiz quadrada média do erro de aproximação = 0,09, e índice de ajuste normalizado = 0,98. Foram encontradas correlações significativas para a intensidade da dor, interferência da dor e humor do paciente (coeficientes de correlação variaram 0,48-0,66, P <0,01). Nas comparações entre grupo controle (pacientes com escores de dor na VAS igual ou inferior a 40 mm na maior parte do dia nos últimos seis meses), e pacientes com condições dolorosas específicas observou-se pontuações mais baixas de catastrofização no grupo controle. No grupo com CTC a relação entre a curva de cortisol salivar, obtida em três pontos do dia (08:00, 16:00 e 22:00 horas) e a catastrofização de acordo com os grupos de catastrofização ( escores B-PCS) estratificados em níveis alto e baixo (alto> Q75 = 42 ou baixo Q75 < 42 ), utilizou-se análise de variância de medidas repetidas (ANOVA), com teste post hoc de Bonferroni. Pacientes com altos escores de catastrofismo apresentaram supressão da secreção de cortisol às 08:00 (p <0,05). Usando modelo multivariado de regressão linear, os fatores correlacionados positivamente com a variável dependente (escores da B-PCS) foram os fatores independentes: níveis séricos de TNF, pontuação no HIT6 e idade (p <0,05). O uso de antidepressivos reduziu em 21% o incremento nos escores da B-PCS. Conclusão: Nossos resultados suportam a validade e confiabilidade da B-PCS. A escala mostrou propriedades psicométricas satisfatórias. A estrutura de três fatores apresentou boas propriedades discriminatórias na comparação de pensamentos catastróficos de sujeitos controles, fibromiálgicos e CTC. A B-PCS mostrou-se instrumento com perfil satisfatório para uso em pesquisa e clínica no Brasil. Também, observamos que a catastrofização está correlacionada com o impacto da CTH, menor oscilação circadiana na secreção de cortisol salivar e níveis séricos de TNF. Isto sugere que o comportamento catastrófico possui substrato biológico que indica sua associação com o estresse crônico e resposta inflamatória. / Theoretical basis: Chronic pain is due to structural and functional changes that influence the maladaptive response to stimuli or processes that underlie excitability. One of the symptoms that permeates large number of patients with chronic pain is the catastrophizing, whose characteristics are a set of negative thoughts, holplessness and magnification of the symptom or condition. This symptom is measured through a catastrophizing scale used in several countries. However, we do not have this instrument for the Portuguese. Objectives: To validate the PCS for Brazil’s Portuguese (B) and verify its psychometric properties. Check the internal consistency, factor structure, and its ability to discriminate patients with specific conditions of chronic pain chronic such as chronic tension type headache (CTH) in accordance with International Headache Society and fibromyalgia according to the criteria of the American College of Rheumatology. To evaluate the possible neurobiological mechanisms correlated with the level of catastrophic symptoms through Cortisol and TNF dosages in a sample of patients with CTH. Methods: 384 subjects aged 18-79 years with chronic musculoskeletal pain participated in this cross-sectional study. The version of the B-PCS was applied as well as pain intensity, pain interference in functional ability, mood and a socio-demographic questionnaire. The discriminatory capacity of B-PCS was assessed in a subsample of patients with chronic tension type headaches (CTH) in accordance with the criteria of the International Headache Society (n = 19), and another with a diagnosis of fibromyalgia according to the criteria of the American College of Rheumatology (n = 50). After the validation the B-PCS was applied in a group of patients with CTH. The impact of headache were evaluated using the Short-Form Headache Impact Test (HIT-6), salivary cortisol samples collected at 08:00, 16:00 and 22:00 and serum TNF. Results: There was good internal consistency [Cronbach's α values of 0.91 for the total PCS-BR. For subdomains 0.93 (holplessness), 0.88 (magnification), 0.86 (rumination)].The coefficients of item-total correlation ranged from .91 to .94. Confirmatory factor analysis supported the three factor structure, with the comparative fit index = 0.98, root mean square error of approximation = 0.09, standard setting and the index of = 0.98. Significant correlations were found for pain intensity, pain interference and mood of the patient (correlation coefficients ranged from .48 to .66, P <0.01). Comparisons between the control group (patients with VAS pain scores at or below 40 mm in most of the day in the last six months), and patients with specific pain conditions were observed lower catastrophizing scores in the control group. In the group with CTH the relationship between the salivary cortisol curve, obtained at three points of the day (08:00, 16:00 and 22:00 hours) and catastrophizing according catastrophizing groups (B-PCS scores) stratified into low and high level (high>Q75=42 or low Q75<42), we used analysis of variance for repeated measures (ANOVA) with Bonferroni post hoc test. Patients with high scores of catastrophism had suppression of cortisol secretion at 08:00 (p <0.05). Using a multivariate linear regression model, the factors positively correlated with the dependent variable (scores of B-PCS) were the independent factors: serum levels of TNF, HIT6 score and age (p <0.05). The use of antidepressants decreased by 21% the increase in scores of B-PCS. Conclusion: Our results support the validity and reliability of the B-PCS. The scale showed satisfactory psychometric properties. The three-factor structure showed good discriminatory properties in comparison to control subjects, fibromyalgia, and HSC catastrophic thoughts. The B-PCS showed to be an instrument with a profile suitable for use in research and clinical practice in Brazil. Also, we found that catastrophizing is correlated with the impact of CTH, lower circadian oscillation in the secretion of salivary cortisol and serum levels of TNF. This suggests that the catastrophic behavior has biological substrate indicating its association with chronic stress and inflammatory response.